Prodefensin-A6 assay method for the in vitro diagnosis of colorectal cancer

ABSTRACT

The present invention relates to a method for the in vitro diagnosis of colorectal cancer by determining the presence of the Prodefensin-A6 tumor marker in a biological sample taken from a patient suspected of having colorectal cancer, it being possible for said method to be used both in early diagnosis, screening, therapeutic follow-up and prognosis, and in relapse diagnosis in relation to colorectal cancer.

This is a divisional of application Ser. No. 13/260,854 filed Sep. 28,2011, which is a National Stage Application of PCT/FR2010/050620 filedApr. 1, 2010, and claims the benefit of French Application No. 0952192filed Apr. 3, 2009. The entire disclosures of the prior applications arehereby incorporated by reference herein in their entirety.

The present invention relates to the cancerology field. Moreparticularly, the subject of the present invention is a method for thein vitro diagnosis of colorectal cancer in a human patient, bydetermining the presence of Prodefensin-A6 in a biological sample takenfrom this patient, it being possible for said method to be used both inearly diagnosis, screening, therapeutic follow-up and prognosis, and inrelapse diagnosis in relation to colorectal cancer.

Colorectal cancer (CRC) is a major public health problem. The worldwideincidence thereof was estimated at 875 000 new cases in 1996¹. Takinginto account both sexes, it is the cancer that occurs most frequently inwestern countries, where it is generally classed among the first 3 mostcommon causes of death due to cancer. The 5-year survival rate, allstages taken into account, is in the region of 60%.

Only early diagnosis offers the hope of a curative treatment. However,at the current time, there is no serological screening test nor specificdiagnostic test which is early.

Screening for colorectal cancer is currently carried out in Europe withtwo distinct approaches: firstly, using a paraclinical test whichconsists in looking for the presence of blood in the stools (FaecalOccult Blood Test, FOBT, marketed, for example, under the nameHemoccult®). This technique has demonstrated its clinical usefulness.When it is used every 2 years in individuals between the ages of 50 and74, it can reduce by 15 to 20% mortality due to colorectal cancer². Forthis, it is necessary for more than half the population concerned toparticipate regularly in the screening and for a colonoscopy to becarried out in the event of a positive test, optionally followed by anappropriate treatment.

Nevertheless, this screening technique suffers from a certain number ofhandicaps:

-   -   The major drawback of this test is its mediocre sensitivity,        most especially for adenomas (precancerous dysplastic lesion)        which, if they are large in size or represent severe dysplasia,        will result in the development of cancer in 1 case out of 10.    -   The test is also not very specific. The appearance of blood in        the stools may be related to a nontumour condition: ulcerative        colitis, haemorrhoids, fistulae, etc. In this case, an        investigation by colonoscopy must be carried out, with the        drawbacks described hereinafter.    -   Finally, Hemoccult® tests are difficult to interpret; they must        therefore be read in specialized centres, by qualified competent        personnel.

Immunological tests specific for human haemoglobin (Feta EIA®, HemeSelect®, etc.) have also been described. They probably constituteprogress compared with Hemoccult®, but they essentially exhibit the sameproblems. Thus, InSure™ marketed by Enterix Inc., makes it possible todetect 87% of patients suffering from CRC and 47% of those havingprecancerous polyps. It is a test for detecting human haemoglobin in thestools, and more particularly the globin portion of this molecule.

A second screening strategy is the systemic performing of a colonoscopyafter the age of 50, which makes it possible in theory to reducemortality due to colorectal cancer. However, the acceptability of thisexamination in individuals who are in good health is too low for ascreening policy using endoscopy to reduce mortality (the level ofcompliancy for colonoscopy in European countries having set up thisscreening strategy is around 2%). There is a not insignificant risk(0.1%) of perforation and bleeding of the colon and of death (1/10 000),and it is also very expensive for public health. Furthermore,colonoscopy requires a very restrictive prior colonic preparation, whichin large part explains the poor compliance.

Tumour markers that can be assayed by immunoassays have for a long timebeen described in the context of colorectal cancer. They are inparticular the carcinoembryonic antigen (CEA) and CA19-9.

CEA is used for follow-up. It cannot be used for the screening or forthe early diagnosis of colorectal cancer because its sensitivity and itsspecificity are insufficient. This is because this marker is expressedby other types of cancer, and in benign pathologies. Despite everything,it is possible to increase sensitivity without losing specificity bycombining, with CEA, another tumour marker such as CA19-9 or CA72-4.

The causes of physiological variations in CA19-9 are rare, but otherbenign conditions (hepatobiliary conditions, pancreatic conditions), ormalignant conditions may induce an increase in CA19-9. This marker,taken alone, is therefore also of no interest for diagnosis.Nevertheless, since its serum concentration is correlated with the sizeof the tumour and the presence of metastases, it may also enable atherapeutic follow-up or the early demonstration of relapses.

Commercially available tests have, moreover, been proposed, such as:

-   -   Colopath®/ColorectAlert^(MD), marketed by Ambrilia, is a rapid        and relatively noninvasive screening test for CRC. Colopath®        detects a plasmalogen (class of complex lipids which are part of        phospholipids) in the rectal mucus of individuals with a        colorectal pathological condition, whereas ColorectAlert^(MD)        detects T-antigen, a complex sugar in the rectal mucus. The        Colopath®/ColorectAlert^(MD) test involves the application of        rectal mucus to a test strip, and the positive or negative        result is based on a Schiff reaction. Ambrilia has studied 1787        individuals and demonstrated that Colopath®/ColorectAlert^(MD)        detects 54% of cases of early-stage colorectal cancer and 49% of        all stages combined.    -   COLARIS, marketed by Myriad Genetics, is a test for detecting,        in the blood, mutations in the MLH1 and MSH2 genes for screening        for hereditary nonpolyposis colon cancer (HNPCC syndrome). The        result of the test is available in 3 weeks. Myriad uses the most        sensitive and most specific sequencing techniques currently        available. The test is expensive.    -   DR-70®, marketed by AMDL, is a test to screen for various types        of cancer (lung, colon, breast, liver, stomach, etc.). It is not        therefore specific for CRC. The principle of said test is based        on the double sandwich ELISA technique (assaying of the DR-70        antigen). Revealing is carried out by enzymatic reaction        (antibodies coupled to biotin and to streptavidin). A coloured        reaction indicates the presence of cancer.

The applicant has now surprisingly demonstrated a novel marker foradenocarcinoma, which is released out of the cancerous tissues by themalignant colonic tumours and is characteristic of these tumours, suchthat it can be detected both in biological samples remote from themalignant tumours, and in the tumours themselves.

Thus, a first subject of the present invention is a method for the invitro diagnosis of colorectal cancer by determining the presence ofProdefensin-A6 in biological samples taken from patients suspected ofhaving colorectal cancer, and preferably remote from the tumours.

The present invention also relates to the use of this method both inearly diagnosis, screening, therapeutic follow-up and prognosis, and inrelapse diagnosis in relation to colorectal cancer.

The method of the invention therefore makes it possible to diagnosecolorectal cancer specifically and early by means of a simple testconsisting in searching for the presence of Prodefensin-A6 in abiological sample taken from a patient, said sample being preferablyremote from the potential tumour. Indeed, the applicant has shown,unexpectedly, that colonic tumours not only specifically secreteProdefensin-A6, but especially release it out of the cancerous tissue,as will be demonstrated in greater detail hereinafter, and that itsconcentration in the biological sample in which the method of theinvention is carried out is increased in comparison with the referencevalues determined for healthy patients.

The determination of the presence of Prodefensin-A6 in a biologicalsample which may or may not be remote from the tumour thus makes itpossible to conclude with respect to the pathological condition sought.One of the advantages of the method of the invention therefore lies inthe possibility of using a sample remote from the potential tumour as adiagnostic sample, thereby enabling a simple and noninvasive diagnosis,whereas a tissue diagnosis requires a biopsy taken invasively. Indeed,the study of tissue markers, for example on a tissue section(immunohistochemistry), may be of prognostic interest, but is of nointerest for screening or diagnosing colorectal cancer.

Defensins are a family of antimicrobial peptides involved in the host'sdefence against microbial attacks. They consist of 30 to 40 amino acidsand have the property of selectively disaggregating membranes. Likeother eukaryotic proteins, Defensins can be present in the form of amature protein or in the form of a precursor.

A precursor, also called precursor protein, consists of a propeptide anda mature part. Thus, Prodefensin-A6 is the precursor protein of themature Defensin-A6 protein, consists of 100 amino acids and comprises asignal peptide (amino acids 1-19), the propeptide (amino acids 20-65)and the mature Defensin-A6 protein (amino acids 66-100).

In general, precursor proteins have for a long time been considered tobe only metabolic molecules. However, a certain number of recentexamples, in particular in the neuropeptide field, indicate that, incertain situations, precursor proteins have a biological activity thatis specific and dissociative from that of the mature peptide that theycan generate. Admittedly, the sequence of precursor proteins includesthat of the mature proteins, for example the sequence of theProdefensins includes that of the Defensins, but their isoelectricpoints and molecular weights are different. Defensins and Prodefensinsare therefore to be considered as two different proteins.

Defensins alpha 5³ and alpha 6⁴ are essentially produced by Paneth cellsof the small intestine. The mRNAs of Defensins alpha 5 and 6 areoverexpressed in colonic tissue in the case of Crohn's disease⁵.Defensin alpha 6 was identified as a potential marker for colon cancerby Nam et al.⁶. Nam et al. developed a competitive ELISA assay whichspecifically assays Defensin alpha 6. They defined a threshold (30ng/ml) beyond which patients were diagnosed as having colorectal cancer.During an analysis of 18 sera from healthy donors and 49 cancer sera,they obtained a diagnostic sensitivity of 69.4% for a diagnosticspecificity of 83.3%. No mention was made in said document regarding theDefensin alpha 6 precursor, Prodefensin alpha 6.

Thus, the Defensin alpha 6 precursor, Prodefensin-A6 (Swiss Prot No.Q01524), has never been described as possibly being of use as a markerin relation to cancer and in particular to colorectal cancer and aspossibly being assayed in a biological sample which may or may not beremote from the malignant tumour.

The expression “determining the presence of the precursor protein” isintended to mean determination of the precursor beyond the referencevalues determined for healthy patients. The precursor sought may be theintact precursor of 100 amino acids, the precursor protein without thesignal peptide (amino acids 20 to 100) or the propeptide alone (aminoacids 20 to 65). It may also be fragments of the latter, such asfragments of the propeptide, with the exclusion of the mature protein(amino acids 66-100) and fragments thereof.

According to one particular embodiment of the invention, the presence ofthe precursor of Prodefensin-A6, without the signal peptide, isdetermined. The sequence described for this precursor in the Swiss-Protdatabase is SEQ ID No. 1(EPLQAEDDPLQAKAYEADAQEQRGANDQDFAVSFAEDASSSLRALGSTRAFTCHCRRSCYSTEYSYGTCTVMGINHRFCCL; corresponding to amino acids 20-100).Preferably, the presence of the propeptide itself having at least thesequence SEQ ID No. 2 (EPLQAEDDPLQAKAYEADAQEQRG ANDQDFAVSFAEDASSSLRALG)and at most the sequence SEQ ID No. 1 is determined.

As is well known to those skilled in the art, protein polymorphismsexist, and the sequences given above are merely indicative. They are theconsensus sequences indicated in the Swiss-Prot database, but amino acidsubstitutions may exist in a percentage that will be evaluated by thoseskilled in the art in order to consider whether it is the same protein.Likewise, the site for cleavage of the propeptide at amino acid 65 istheoretical and is given only by way of indication.

The expression “release by colonic tumours” is intended to mean theactive or passive secretion or the release, whatever the mechanism, ofthe tumour marker by the tumour cells themselves or by the neighbouringnontumour cells following lesions or modifications of cell phenotyperesulting from the tumour development.

The expression “biological sample in which the method of the inventionis carried out” is intended to mean any biological sample which iscapable of containing the tumour marker of interest. By way of exampleof a biological sample not remote from the tumour, mention may be madeof solid samples such as the tissue originated from the tumour, frombiopsies of this tumour, from lymph nodes, from the patient'smetastases, and the cells purified from these solid samples. By way ofexample of the biological sample remote from the tumour, mention may bemade of biological fluids such as whole blood or derivatives thereof,for example serum or plasma, urine, saliva and effusions, bone marrowand stools, and the cells purified from these liquid samples. Blood orderivatives thereof and also stools, effusions and cells purified fromthese liquid samples are preferred.

The method of the invention may be improved by detecting, in addition tothe Prodefensin-A6, at least one other tumour marker, where appropriatealso released out of the cancerous tissues by the colonic tumours. Thus,the combination of at least two markers makes it possible to improve thespecificity and the sensitivity of the diagnostic test for colorectalcancer.

Thus, another subject of the invention also consists in determining thepresence of at least one other tumour marker chosen from the group offollowing markers: leukocyte elastase inhibitor, ezrin, aminoacylase 1,liver fatty acid-binding protein, intestinal fatty acid-binding protein,apolipoprotein AI, apolipoprotein AII, I-plastin, beta2-microglobulin,proteasome 20S, galectin-3, L-lactate dehydrogenase chain B,calreticulin, regenerating islet-derived protein 3 alpha,tumour-associated calcium signal transducer 1, keratin type IIcytoskeletal 8, keratin type I cytoskeletal 18, keratin type Icytoskeletal 19, epithelial cadherin, CEA, villin, CA19-9, CA 242, CA50, CA 72-2, testosterone, TIMP-1, cripto-1, protein disulphideisomerase, intelectin-1, cytokeratin 20, translationally-controlledtumour protein, (Pro)defensin-A5, MIF, pyruvate kinase M2-PK,calgranulin C, CD24, CCSA-3 (colon cancer specific antigen) and CCSA-4,the detection of DNA fragments in the blood having specific alterationsto their methylation profile, for instance methylated DNA of the AXL4gene (aristaless-like homeobox-4 gene methylation) or the methylated DNAof the septin-9 gene, the detection of specific alterations in faecalDNA fragments, such as specific mutations of faecal DNA or specificalterations of the methylation profile of faecal DNA, the detection ofhuman faecal haemoglobin.

The expression “tumour marker other than Prodefensin-A6” is intended tomean the protein, the messenger RNA or specific modifications of thecorresponding gene, such as mutations or methylations. In other words,only Prodefensin-A6 is solely sought in the form of a protein, which maybe complete or in the form of a fragment.

The leukocyte elastase inhibitor tumour marker (Swiss Prot No. P30740,also known as LEI, serpin B1, monocyte/neutrophil elastase inhibitor,M/NEI or EI) was sequenced in 1992⁷. LEI specifically inhibits proteaseshaving elastase-type or chymotrypsin-type properties by formation of thecomplex that cannot be dissociated under the action of SDS⁸. LEI thusinhibits three of the major proteases produced by neutrophils: leukocyteelastase, proteinase-3 and cathepsin G. These proteases enable theimmune system to defend the organism by proteolysis of extracellular orphagocytosed substrates. However, when these proteases are in excess,they are responsible for inflammatory reactions. LEI could thereforehave a role in regulating and limiting the inflammatory action inducedby cell proteases. The applicant has shown, for its part, surprisingly,in patent application WO2009/024691, that the concentration of thisprotein is increased relative to the reference values determined forhealthy patients, such that this protein is a good marker in biologicalsamples taken from a patient having colorectal cancer, said samplesbeing possibly remote from the tumour.

The ezrin marker (Swiss Prot No. P15311, also known as p81, cytovillinor villin-2) is a protein which provides binding between the cellmembrane and the actin filaments of the cytoskeleton of the cell, inparticular in the microvilli of intestinal epithelial cells⁹. W. G.Jiang and S. Hiscox¹⁰ have shown that the interleukins IL-2, IL-8,IL-10, etc., can inhibit the expression of ezrin in the HT29 humancolorectal cancer cell line. The same authors¹¹ have shown that theinhibition of ezrin expression in the HT115 and HRT18 colorectal cancercell lines reduces the adhesion between cells and increases the mobilityand the invasive behaviour of the cells. They have concluded that ezrinregulates cell/cell and cell/matrix adhesions by interacting with thecell adhesion molecules E-cadherin and beta-catenin. They have suggestedthat ezrin could play an important role in controlling the invasivepotential of cancer cells. Moreover, T. Xiao et al.¹² have used an ELISAassay to quantify the plasma ezrin of patients with lung cancer.However, they have not observed any differences compared with controlindividuals. The applicant has shown, for its part, surprisingly, inpatent application WO2009/019365, that the concentration of this proteinis increased relative to the reference values determined for healthypatients, such that this protein is a good marker in biological samplestaken from a patient having colorectal cancer, said samples possiblybeing remote from the tumour.

The aminoacylase 1 marker (Swiss Prot No. Q03154, also known as EC3.5.1.14, N-acyl-L-amino acid amidohydrolase or ACY-1) is part of theaminoacylase family. They are enzymes which catalyse the hydrolysis ofacylated amino acids so as to give fatty acids and amino acids¹³. Animmunochemical assay for aminoacylase enzymatic activity was developedas early as 1975 by K. Lorentz et al.¹⁴ and was used to assay varioustissues and sera¹⁵. The study showed an increase in aminoacylaseactivity in the case of hepatic pathological conditions but not in thecase of colon cancer. Moreover, the aminoacylase 1 gene has beenidentified on chromosome 3p21.1¹⁶. The 3p21.1 region is reduced tohomozygocity in small cell lung cancer, and in this case, theaminoacylase expression is repressed or undetectable¹⁷. Similarly, S.Balabanov et al.¹⁸ have shown that the aminoacylase expression isrepressed in the case of kidney cancer. The applicant has shown, for itspart, surprisingly, in patent application WO2009/019366, that theconcentration of this protein is increased relative to the referencevalues determined for healthy patients, such that this protein is a goodmarker in biological samples taken from a patient having colorectalcancer, said samples possibly being remote from the tumour.

The liver fatty acid-binding protein marker (Swiss Prot No. P07148, alsoknown as L-FABP, FABP1, FABPL, Z-protein or sterol transporter protein)belongs to the FABP family which comprises nine isoforms. Each isoformis named according to the tissue in which it was first detected. Theseisoforms have a shared function and similar three-dimensionalstructures, but their sequence homology is not high. L-FABP wassequenced in 1985¹⁹. It is a small protein of 15 kDa that is abundant inthe cytosol and has the ability to bind to free fatty acids and also tobilirubin. Some recent studies appear to indicate that impairments inexpression of the L-FABP protein could induce a tumorigenesis process.For prostate cancer, the level of expression of L-FABP mRNAs in tumourtissue biopsies was ten times higher than in the normal tissue²⁰. Forcolon cancer, several teams have identified a decrease in the expressionof L-FABP protein in the tumour tissue compared with normal colonicmucosa, using two-dimensional electrophoresis techniques²¹. This resulthas also been confirmed by immunohistochemistry techniques. In addition,the L-FABP protein is a prognostic liver resection marker in patientswith colorectal cancer having metastasized to the liver²². In patentapplication WO00/33083, it has been suggested that this marker could bedetected in biological fluids from patients having colon cancer. Theapplicant has, for its part, confirmed, in patent applicationWO2009/019368, that the concentration of this protein is decreasedrelative to the reference values determined for healthy patients, suchthat this protein is a good marker in biological samples taken from apatient having colorectal cancer, said samples being remote from thetumour.

The intestinal fatty acid-binding protein marker (Swiss Prot No. P12104,also known as I-FABP, FABP-2 or FABPI) was sequenced in 1987²³. It is asmall protein of 15 kDa that is abundant in the cytosol and that has theability to bind to free fatty acids and also to bilirubin. The I-FABPprotein is expressed in the enterocytes of the small intestine and canconstitute approximately 2% of the protein content of this cell type. Atthe tissue level, the duodenum and the jejunum contain significantlyhigher amounts of I-FABP than the colon (jejunum: 4.8 μg/g, colon: 0.25μg/g)²⁴. I-FABP could not be detected in the plasma samples of healthyindividuals. On the other hand, in certain pathological contexts such asintestinal ischaemia, Crohn's disease or primary biliary cirrhosis, itis possible to demonstrate an increase in the plasma I-FABPconcentration in certain individuals²⁴. For prostate cancer, it has beenshown that the level of expression of I-FABP mRNA in biopsies of tumourtissue is seven times higher than in normal tissue²⁰. In the model ofinduction of a colorectal tumour with azoxymethane in rats, the level ofexpression of I-FABP mRNA is reduced by 2.92 to 3.97 times when theanimals have a diet that reduces the incidence of cancer (soya proteinsor whey hydrolysate)²⁵. The applicant has confirmed, for example inpatent application WO2009/019366, that the concentration of this proteinis increased relative to the reference values determined for healthypatients, such that this protein is a good marker in biological samplestaken from a patient having colorectal cancer, said samples being remotefrom the tumour.

Apolipoproteins are a family of proteins consisting of polar amino acidsenabling the transport of lipids in the blood through the formation of ahydrophilic macromolecular complex called a lipoprotein. For each of thehuman plasma apolipoproteins there are isoforms derived from geneticpolymorphism and/or from post-translational modifications, the presenceof which in the blood can be associated with certain pathologicalconditions²⁶. The plasma concentration of apolipoproteins is notinsignificant, of the order of 1 mg/ml²⁷.

The apolipoprotein AI marker (NCBI No. 490098, also known as Apo A-I,Apo AI and Apo A1) is a protein of 243 amino acids and of 28 kDa. It isessentially synthesized by the liver and the intestine. This protein hasbeen shown to be underabundant in the sera of patients suffering fromcolorectal cancer compared with healthy individuals, by SELDI-TOF²⁸.However, it is specified in this article that patients with CRC aredistinguished from healthy individuals by combining Apo AI with otherprotein markers. Moreover, this article specifies that the assaying ofApo AI by turbidimetric immunoassay, carried by another team, does notconfirm the underabundance of this protein in the sera of patientshaving CRC²⁹. Hachem et al.³⁰ have, for their part, assayed Apo AI insera of patients having had liver cancer following colorectal cancermetastases. The applicant has shown, for its part, surprisingly, thatassaying by immunoassay makes it possible to demonstrate a decrease inthe concentration of this protein in patients having colorectal cancer,contrary to what was put forward by Engwegen et al.²⁸, who were able todemonstrate this decrease only by implementing the SELDI-TOF technique.The assaying of Apo AI by immunoassay in biological samples is a goodmethod for the diagnosis of colorectal cancer, said samples being remotefrom the tumour, insofar as the assaying by immunoassay that is carriedout is not turbidimetry as used by the team of Zhang et al.²⁹.

The apolipoprotein AII marker (Swiss Prot No. P02652, also known as ApoAII, Apo-AII and Apo A2) is a protein of 17 380 Da composed of twopolypeptide chains of 77 amino acids each, linked by a disulphidebridge. Like apolipoprotein AI, apolipoprotein AII is essentiallysynthesized by the liver and the intestine. Hachem et al.³⁰ have alsoassayed, in addition to Apo AI, the Apo AII in sera of patients havinghad liver cancer following colorectal cancer metastases. However, theresults are not significant and do not enable a conclusion to be drawnas to the pathological condition sought. The applicant has shown, forits part, surprisingly, in patent application WO2009/019370, that theconcentration of this protein is decreased relative to the referencevalues determined for healthy patients, such that the decrease in theconcentration of this protein in patients having colorectal cancer makesit a good marker in biological samples taken from a patient havingcolorectal cancer, said samples being remote from the tumour.

The I-plastin marker (Swiss Prot No. Q14651, also known asintestine-specific plastin or plastin 1) belongs to the family of humanplastins of which three representatives are known: I-plastin, L-plastinand T-plastin. Some authors call plastins “fimbrins”, yet other authorsreserve the name fimbrin for I-plastin. The plastins are proteins thatbind to actin so as to form the cytoskeleton (cell skeleton). They are70 kDa proteins that are relatively well-conserved throughout eukaryoticevolution. They exhibit strong tissue specificity, only one isoform at atime is present in normal tissues³¹. The use of plastins with respect tocancer has already been described in U.S. Pat. No. 5,360,715, whichproposes a method for determining whether a cell is haematopoietic orneoplastic, i.e. cancerous. This method claims the assaying of L-plastinand of T-plastin at the cellular level, and more particularly theassaying of the mRNA thereof. However, despite these properties, noprior study has been carried out to evaluate the importance of plastinsin relation to the diagnosis of colorectal cancer using a serum orfaecal sample. Furthermore, I-plastin has never been envisaged as apotential cancer marker³². The applicant has shown, for its part,surprisingly, in patent application WO2009/019369, that theconcentration of this protein is increased relative to the referencevalues determined for healthy patients, such that this protein is a goodmarker in biological samples taken from a patient having colorectalcancer, said samples possibly being remote from the tumour.

The beta2-microglobulin marker (Swiss Prot No. P61769, also known asβ2-microglobulin, β2M) is a low-molecular-weight protein (11 to 12 kDa)found at the surface of most nucleated human cells. The serumβ2-microglobulin level increases in certain patients suffering fromcancer, without this increase being specific, or correlated with thenature of the tumour, its stage or the severity of the disease. Asignificant increase is also observed in other diseases, such as lupuserythematosus, rheumatoid arthritis, Sjögren's syndrome, malignantdiseases of the lymphoid system (multiple myeloma, B-cell lymphoma),certain viral diseases (hepatitis or AIDS) and in haemophiliac patients.Since β2-microglobulin is filtered by the renal glomeruli and reabsorbedby the proximal convoluted tubules, its concentration in the blood maybe modified in the case of renal pathological conditions. The assayingof β2-microglobulin is thus most commonly reserved for the diagnosis ofrenal pathological conditions, or for the follow-up of infection withthe acquired immunodeficiency virus. However, this marker is known as atumour marker, in particular for colon cancer.

The proteasome 20S marker (also known as prosome) is the centralstructure of the proteasome, which is itself a molecular complexresponsible for the intracellular degradation of ubiquinated proteins³³.The proteasome is a molecular complex of 700 kDa, consisting of 28subunits associated in four rings of seven subunits. In humans, sevenalpha units (α1, α2, α3, α4, α5, α6 and α7) and ten beta units (β1, β2,β3, β4, β5, β6, β7, β1i, β2i and β5i) are known. By virtue of itscatalytic properties, the proteasome plays a central role in themechanisms of cell proliferation, growth, regulation and apoptosis, andtherefore in the cancerization pathways. Proteasome inhibition withBortezomib (Velcade) is a recognized treatment for multiple myeloma.Phase II or III therapeutic trials are ongoing for haematologicalcancers or tumours. Lavabre-Bertrand et al.³⁴ have shown that the serumlevel of proteasome can be elevated on the occasion of certainpathological conditions, in particular in the case of cancers (myeloma,lymphoma and solid tumours).

The galectin-3 marker (Swiss Prot No. P17931, also known as Gal-3,galactose-specific lectin 3, MAC-2 antigen, IgE-binding protein, 35 kDalectin, carbohydrate binding protein 35, CBP 35, laminin-bindingprotein, lectin L-29, L-31, galactoside-binding protein or GALBP) is alectin capable of binding to beta-galactosidase structures ofN-acetyllactosamine type. It is a protein with multiple functionsinvolved in various biological functions, including the adhesion oftumour cells, proliferation, differentiation, angiogenesis, apoptosisand metastatic cancer progression³⁵. Various studies have shown thatGal-3 can form complexes with numerous molecules: CEA, IgE, laminin,mucin, Mac-2BP, LAMP1, LAMP2, fibronectin, etc. A serum assay of Gal-3has been described by lurisci et al.³⁶. Gal-3 was captured onmicroplates coated with Mac-2-binding protein (a Gal-3-binding protein)and then revealed with an anti-Gal-3 rat antibody. This study showedelevated serum Gal-3 levels in the case of gastrointestinal cancer,breast cancer, lung cancer, ovarian cancer, melanomas and non-Hodgkin'slymphomas.

The L-lactate dehydrogenase chain B marker (Swiss Prot No. P07195, alsoknown as LDH-B, LDH heart unit or LDH-H) is a protein that can formcomplexes in order to form a homotetramer. This protein can also formcomplexes with the L-lactate dehydrogenase chain A protein (Swiss ProtNo. P00338, also known as LDH-A, LDH muscle unit or LDH-M) in the formof heterotetramers. The serum level and/or the serum enzymatic activityof the tetrameric complexes, called LDH, increase(s) in the blood streamproportionally to the tumour mass for many solid tumours. Its use isrecommended in combination with human chorionic gonadotrophin (beta-hCG)and placental alkaline phosphatase for the follow-up of seminal vesiclecancers. LDH is considered to be a marker of interest for the prognosisof lymphomas, of leukaemia and of colon cancer³⁷.

The calreticulin marker (Swiss Prot No. P27797, also known as CRP55,calregulin, HACBP, ERp60 or grp60) is a multifunctional protein. It is alectin capable of interacting transiently with virtually all themonoglycosylated proteins of the endoplasmic reticulum. McCool et al.³⁸have thus shown that calreticulin is involved in maturation of thecolonic mucin MUC2. A method for the diagnosis of CRC which usesassaying of calreticulin in a tissue, the stools or a body fluid isdescribed in patent application WO03/065003.

The regenerating islet-derived protein 3 alpha marker (Swiss Prot No.Q06141, also known as Reg III-alpha, pancreatitis-associated protein 1or pancreatis associated protein I (PAP 1)) is a protein that is weaklyexpressed in the healthy pancreas. It is overexpressed during the acutephases of pancreatitis and in certain patients suffering from chronicpancreatitis. In this case, it appears in the pancreatic fluid and inthe bloodstream³⁹. Motoo et al.⁴⁰ have shown, by ELISA assay, that thelevel of PAP 1 in the blood increases in certain patients having coloncancer, stomach cancer, liver cancer or pancreatic cancer, and also inthe case of renal insufficiency. To do this, they use the ELISA assay(PANCEPAP) sold by the company Dynabio (La Gaude, France).

The tumour associated calcium signal transducer 1 marker (Swiss Prot No.P16422, also known as major gastrointestinal tumour-associated proteinGA733-2, epithelial cell surface antigen, EpCAM, epithelialglycoprotein, EGP, adenocarcinoma-associated antigen, KSA, KS 1/4antigen, cell surface glycoprotein Trop-1 or CD326 antigen) wascharacterized in 1979 by virtue of its ability to be recognized by anantibody directed against colorectal cancer cells⁴¹. This protein isknown by various names, as indicated above, but the most common use isto call it EpCAM. It is a transmembrane protein expressed on thebasolateral surface of cells, in certain epithelia and many cancers⁴².As early as 1982 Herlyn et al.⁴³ showed that the injection of ananti-EpCAM monoclonal antibody could inhibit tumour growth in patientshaving colorectal cancer. These results led to the development of anantitumour treatment based on an anti-EpCAM antibody called Edrecolomab.This treatment is marketed under the name Panorex™. Moreover, Abe etal.⁴⁴ have shown, by ELISA assay, that a soluble form of EpCAM, calledMK-1, is increased in the blood stream by 10% in the cancer patientsstudied.

The cytokeratins are part of the proteins that make up the intermediatefilaments of the cytoskeleton of epithelial cells. Currently, more than20 human cytokeratins have been identified. The cytokeratins 8 (SwissProt No. P05787, also known as cytokeratin-8, CK-8, keratin-8 or K8), 18(Swiss Prot No. P05783, also known as cytokeratin-18, CK-18, keratin-18or K18) and 19 (Swiss Prot No. P08727, also known as cytokeratin-19,CK-19, keratin-19 or K19) are the most abundant in epithelial cells andare useful tools for the diagnosis of cancer pathological conditions⁴⁵.This clinical importance is linked to the release of cytokeratins byepithelial cells in the apoptotic or proliferation phase. In the case ofapoptosis, this release occurs in the form of soluble fragments whichseem to appear under the proteolytic action of caspases. Undegradedcytokeratin forms have never been described in the bloodstream. Thethree cytokeratin assays most commonly used clinically are the tissuepolypeptide antigen (TPA) assay, the tissue polypeptide specific antigen(TPS) assay and the CYFRA 21-1 assay. TPA is a broad-spectrum test whichmeasures cytokeratins 8, 18 and 19. The TPS and CYFRA 21-1 assays aremore specific and measure, specifically, fragments of cytokeratin 18 andof cytokeratin 19. These three assays detect soluble cytokeratinfragments that may be present on their own or in the form of proteincomplexes. TPA, TPS or CYFRA-21-1 have been used for the therapeuticfollow-up of colorectal cancers, breast cancers, lung cancers, bladdercancers, ovarian cancers, pancreatic cancers, prostate cancers andcertain ENT cancers. The assaying of soluble cytokeratin fragments inthe blood in fact has a clinical value in screening for relapses orevaluating the response to the therapy used (radiotherapy, chemotherapy,hormone treatment). Regular assaying makes it possible in particular toevaluate the progression of the tumour mass. The amount of soluble bloodcytokeratins also has a prognostic aspect with respect to the tumourstage and to the formation of metastases. Currently, the blood assay forcytokeratin most commonly used is CYFRA 21-1. It is highly recommendedfor the follow-up of patients having non-small cell lung cancer. Variouscommercially available assays exist for TPA (AB Sangtec Medical Co.,Byk-Roland, etc.), TPS (IDL Biotech AB, BEKI Diagnostics, etc.) andCYFRA-21-1 (Roche Diagnostics, CIS Bio-International, FujirebioDiagnostics, etc.). Moreover, Kim et al.⁴⁶ have shown that assayingfaecal cytokeratin 19 (DiNonA Inc.) may be useful in screening forgastrointestinal diseases, in combination with a faecal occult bloodassay.

The epithelial cadherin marker (Swiss Prot No. P12830, also known asE-cadherin, uvomorulin, cadherin-1, CAM 120/80 or CD324 antigen) is atransmembrane protein that mediates calcium-dependent cell adhesion. Itis specifically expressed in epithelial cells, where it is involved inmaintaining their phenotype. The cytoplasmic domain of E-cadherin bindsto β-catenin, which is itself bound to the actin filament networks ofthe cytoskeleton. This E-cadherin/β-catenin binding plays an essentialrole in stabilizing cell/cell adhesions of the epithelial tissue. Theloss of E-cadherin can therefore reduce the cell adhesion and increasethe invasive capacity of cancer cells. A reduction in expression ofE-cadherin or in β-catenin is generally associated with greateraggressiveness and dedifferentiation of the tumour, in particular forgastrointestinal cancers. Roca et al.⁴⁷ have thus shown that patientshaving colorectal cancer and underexpressing E-cadherin have a moreunfavourable prognosis than patients having a normal expression level.As early as 1983, Damsky et al.⁴⁸ showed that a soluble form ofE-Cadherin could be released by the MCF-7 breast cancer cell line. Thissoluble form corresponds to the cleavage of the extracellular portion ofE-cadherin. Later, Katayama et al.⁴⁹ showed that the soluble form ofE-cadherin could be released into the bloodstream in the case of cancer,and Willmanns et al.⁵⁰ showed that the increase in the amount ofE-cadherin in the blood was correlated with the tumour stage incolorectal cancers. A commercial kit is, moreover, proposed by thecompany Takara BioChemicals (Tokyo, Japan).

The assaying of CEA (carinoembryonic antigen) for the diagnosis ofcolorectal cancer has been proposed since 1965 by Gold and Freedman⁵¹,but a blood assay for this marker has poor sensitivity for the diagnosisof colorectal cancers at a relatively nonadvanced stage. The assaying ofserum CEA is thus especially recommended for evaluating the risk ofliver metastases⁵² and for therapeutic follow-up. In addition, it is amarker that is not very specific for colorectal cancer; it may in factbe increased in many other cancers (lung, breast, etc.). On the otherhand, the assaying of faecal CEA appears to be more sensitive and morespecific than the assaying of serum CEA or than the assaying of faecalblood⁵³. However, this assaying is not yet proposed routinely.

The reactive antigenic determinants 1116-NS-19-9, more commonly calledCA19-9 (carbohydrate antigen 19.9), are carried by high-molecular-weightproteins⁵⁴. The assaying of CA 19-9 in the blood is more specific thanthat of CEA. The CA 19-9 level in the blood increases in the event ofcolorectal cancer, of pancreatic cancer and of liver cancer(cholangiocarcinoma), but also in the event of noncancerous pathologicalconditions (cholangitis, etc.). Its use in combination with CEA isrecommended both at the time of diagnosis of a cancer and for follow-upof the pathological condition.

J. Holmgren et al.⁵⁵ have shown that the amount of CA 50 antigen in theserum is increased in the case of colorectal cancer. The CA 50 antigenis defined by its ability to be recognized by a specific monoclonalantibody.

As regards the CA 72 marker, T. L. Klug et al.⁵⁶ have shown that theamount of CA 72 antigen in the serum is increased in the case ofcolorectal cancer. The CA 72 antigen is defined by its ability to berecognized by a specific monoclonal antibody.

Similarly, P. Kuusela et al.⁵⁷ have shown that the amount of CA 242antigen in the serum is increased in the case of colorectal cancer. TheCA 242 antigen is defined by its ability to be recognized by a specificmonoclonal antibody.

The assaying of testosterone for the diagnosis of colorectal cancer hasbeen proposed in men by M. Holland et al.⁵⁸. These authors have shown afall in the blood testosterone level in the case of colorectal cancer.

As regards the TIMP-1, or tissue inhibitor of matrix metalloproteinasetype-1 marker, patent application US 2007/0020707 describes inparticular the assaying of TIMP-1 for the diagnosis of colorectal cancerby assaying in a body fluid.

F. Model et al.⁵⁹ showed, in July 2006, during the World Congress onGastrointestinal Cancer, that it was possible to detect methylated formsof the septin-9 gene in the plasma of patients having colorectal cancer.

M. P. Ebert et al.⁶⁰ have shown that the ALX4 gene, or aristaless-likehomeobox-4 gene, is more often methylated in the sera of patients havingcolorectal cancer than in control sera (P<0.0001). Using a thresholdvalue of 41.4 pg/ml, they have obtained a sensitivity of 83.3% and aspecificity of 70%.

Villin is described as a blood marker for the diagnosis of colorectalcancer in patent application FR2581456.

C. Bianco et al.⁶¹ have shown that the amount of cripto-1 in the serumis increased in the case of colorectal cancer.

The induction of intestinal tumorigenesis by macrophage migrationinhibitory factor (MIF) has been described by Wilson et al.⁶². Morerecently, it has also been shown, by Lee et al.⁶³, that MIF is apotential blood marker for the early diagnosis of colorectal cancer.

The protein disulphide isomerase marker (Swiss Prot No. P07237, alsoknown as EC 5.3.4.1, PDI, prolyl 4-hydroxylase subunit beta, cellularthyroid hormone-binding protein or p55) is a multifunctional proteinwhich catalyses the formation, the breaking and the rearrangement ofintramolecular disulphide bridges. At the surface of cells, it acts as areductase and cleaves the disulphide bridges of the proteins attached tothe cells. Inside these cells, it is a soluble molecule located in thelumen of the endoplastic reticulum, where it forms and rearranges thedisulphide bridges of neosynthesized proteins. It comprises twothioredoxin-type catalytic domains having a characteristic CXXC motif.At high concentration, PDI functions as a chaperone protein whichinhibits the aggregation of incorrectly folded proteins. At lowconcentration, it has an antagonistic role and facilitates theaggregation. PDI also forms the structural subunit of various enzymes,such as prolyl hydroxylase which catalyses the hydroxylation of theproline residues of the pro-alpha chains of procollagen. In patentapplication EP1724586, PDI has been described as a diagnostic marker forcertain cancers, such as colon cancer.

The assaying of intelectin-1 (Swiss Prot No. Q8WWA0, also known asintestinal lactoferrin receptor, galactofuranose-binding lectin,endothelial lectin HL-1 or omentin) for the diagnosis of colorectalcancer has been described in patent application US2003/0082533.

The use of translationally-controlled tumour protein (Swiss Prot No.P13693, also known as TCTP, p23, histamine-releasing factor, HRF orfortilin) and of Prodefensin-A5 (Swiss Prot No, Q01523) as markers incolorectal cancer is described, respectively, in patent applicationsUS2003/0172388 and US2006/0179496. The term “(Pro)defensin” is intendedto mean the precursor, namely the Prodefensin before cleavage, thepropeptide, namely the N-terminal moiety after cleavage of Prodefensin,and the mature protein, namely the Defensin, corresponding to theC-terminal moiety after cleavage.

M2-PK is an isoenzyme of pyruvate kinase which is found in dimeric ortetrameric form. The dimeric form is predominant in tumour cells and,for this reason, is called tumour M2-PK. Numerous studies have used theassaying of faecal M2-PK by ELISA as a colorectal cancer marker, forinstance Hardt et al.⁶⁴

The use of calgranulin C or S100 A12 protein as a marker for colorectalcancer is described in patent application WO2007/134779.

Sagiv et al.⁶⁵ have shown an increase in the expression of CD24 in thecase of colorectal cancer.

The colon cancer specific antigen (CCSA)-3 and -4 proteins are serummarkers which have also been associated with colorectal cancer by Lemanet al.⁶⁶

Finally, the assaying of human faecal haemoglobin is known practice andcan be implemented as previously described.

The concentration of the tumour marker other than Prodefensin-A6 will,depending on the marker under consideration, be increased or decreasedin the biological sample in which the method of the invention is carriedout, relative to the reference values determined for healthy patients.

Preferably, the tumour marker(s) other than Prodefensin-A6 is (are)chosen from: leukocyte elastase inhibitor, ezrin, aminoacylase 1, liverfatty acid-binding protein, intestinal fatty acid-binding protein,apolipoprotein AI, apolipoprotein AII, I-plastin, protein disulphideisomerase, intelectin-1, cytokeratin 20, translationally-controlledtumour protein, (Pro)defensin-A5, galectin-3, beta2-microglobulin, CEA,CA19-9, TIMP-1, M2-PK and MIF.

More preferably, the tumour marker(s) other than Prodefensin-A6 is (are)chosen from the markers: L-FABP, beta2-microglobulin, galectin-3, CEA,CA19-9, MIF and I-plastin.

According to one particular embodiment, the method of the inventioncomprises or consists of the detection of the following markers:

-   -   Prodefensin-A6 and L-FABP,    -   Prodefensin-A6, CA19-9 and CEA,    -   Prodefensin-A6, beta2-microglobulin and CEA,    -   Prodefensin-A6, beta2-microglobulin, CA19-9 and CEA,

Prodefensin-A6, beta2-microglobulin, L-FABP and CEA,

-   -   Prodefensin-A6, L-FABP, CA 19-9 and CEA,    -   Prodefensin-A6, beta2-microglobulin, CA19-9 and CEA,    -   Prodefensin-A6, beta2-microglobulin, CA19-9, L-FABP and CEA,    -   Prodefensin-A6, beta2-microglobulin, CA19-9, galectin-3, L-FABP,        MIF and CEA,    -   Prodefensin-A6, beta2-microglobulin, CA19-9, galectin-3, L-FABP,        MIF, I-plastin and CEA.

Of course, the method of the invention may also include the detection ofany other colorectal cancer marker known to those skilled in the art.

As indicated previously, the tumour marker(s) of interest is (are)detected either in the form of protein, or in the form of messenger RNA,or by modification of the corresponding DNA (mutation or modification ofmethylations), it being understood that Prodefensin-A6 is detected onlyin the form of protein, which may be whole protein or in the form of aprotein fragment.

The determination of the presence, in the biological sample, of the“protein” tumour marker of interest can be carried out by any method fordetermining the presence of a protein in a sample, known to thoseskilled in the art, such as, for example a biochemical test, includingan immunoassay, or by mass spectrometry.

The biochemical test may be any test widely known to those skilled inthe art involving molecular intereactions, i.e. reactions between saidtumour marker and one or more binding partner(s) specific or notspecific for said tumour marker.

Preferably, the biochemical test is an immunoassay known to thoseskilled in the art, involving immunological reactions between the tumourmarker, which is the antigen, and one or more specific bindingpartner(s), namely the antibodies directed against this antigen.

The binding partners specific or not specific for the tumour marker(s)sought in the method of the invention are any partner capable of bindingto this or these marker(s). They are said to be specific when they arecapable of binding to these markers with a high specificity, or even aspecificity of 100%. They are said to be nonspecific when theirspecificity of binding to these markers is low and they are then capableof binding to other ligands, such as proteins. By way of example,mention may be made of antibodies, antibody fractions, receptors and anyother molecule capable of binding to this marker.

The binding-partner antibodies are, for example, either polyclonalantibodies or monoclonal antibodies.

According to one particular embodiment, the method of the invention usesa binding partner specific for Prodefensin-A6, of sequence SEQ ID No. 2.Preferably, the binding partner specific for Prodefensin-A6 is amonoclonal antibody which recognizes any linear or conformationalepitope included in the sequence SEQ ID No. 2.

Preferably, the monoclonal antibody specifically recognizes a linearepitope of at least the sequence DP (SEQ ID No. 4), preferably of atleast the sequence EDDPLD (SEQ ID No. 6) and of at most the sequence SEQID No. 2, or else it specifically recognizes a conformational epitopechosen from the following epitopes:

-   -   an epitope of at least sequence X₁X₂X₃X₄X₅X₆X₇X₈R (SEQ ID No.        7), in which X₁ is V or L, X₂ is T or L, X₃ is P, S or C, X₄ is        P or S, X₅ is W or T, X₆ is A, Q, M, C or E, X₇ is I, E or D and        X₈ is F, Y, S or L, and of at most the sequence SEQ ID No. 8,        SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 12, as        indicated in FIG. 4,    -   an epitope of at least the sequence X₁X₂X₃X₄X₅X₆HX₇ (SEQ ID No.        13), in which X₁ is S or T, X₂ is C or absent, X₃ is T, L or E,        X₄ is H or R, X₅ is I, F or E, X₆ is G or V and X₇ is C or N,        and of at most the sequence SEQ ID No. 14, SEQ ID No. 15 or SEQ        ID No. 16, as indicated in FIG. 4,    -   an epitope of at least the sequence X₁HPX₂X₃X₄X₅X₆X₇ (SEQ ID No.        17), in which X₁ is P or W, X₂ is W or E, X₃ is S, A, Q or W, X₄        is M, L, R or P, X₅ is H, F, W or G, X₆ 1 S V or A and X₇ is I        or V, and of at most the sequence SEQ ID No. 18, SEQ ID No. 19,        SEQ ID No. 20 or SEQ ID No. 21, as indicated in FIG. 4,    -   an epitope of at least the sequence X₁HX₂X₃X₄X₅ (SEQ ID No. 22),        in which X₁ is Y or N, X₂ is E, D or Q, X₃ is T, N, R, M or K,        X₄ is W, H or F and X₅ is P or G, and of at most the sequence        SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ        ID No. 27, SEQ ID No. 28 or SEQ ID No. 29, as indicated in FIG.        4.

The monoclonal antibodies which specifically recognize the propeptidepart of Prodefensin-A6 are novel and constitute another subject of theinvention.

According to one embodiment, the monoclonal antibodies of the inventionspecifically recognize an epitope having at least the sequence SEQ IDNo. 4, preferably at least the sequence SEQ ID No. 6 (epitope 1), and atmost the sequence SEQ ID No. 2.

According to another embodiment, the anti-Prodefensin-A6 monoclonalantibodies of the invention specifically recognize an epitope chosenfrom the following epitopes:

-   -   an epitope of at least the sequence SEQ ID No. 7, in which X₁ is        V or L, X₂ is T or L, X₃ is P, S or C, X₄ is P or S, X₅ is W or        T, X₆ is A, Q, M, C or E, X₇ is I, E or D and X₈ is F, Y, S or        L, and of at most the sequence SEQ ID No. 8, SEQ ID No. 9, SEQ        ID No. 10, SEQ ID No. 11 or SEQ ID No. 12 (epitope 2),    -   an epitope of at least the sequence SEQ ID No. 13, in which X₁        is S or T, X₂ is C or absent, X₃ is T, L or E, X₄ is H or R, X₅        is I, F or E, X₆ is G or V and X₇ is C or N, and of at most the        sequence SEQ ID No. 14, SEQ ID No. 15 or SEQ ID No. 16 (epitope        3),    -   an epitope of at least the sequence SEQ ID No. 17, in which X₁        is P or W, X₂ is W or E, X₃ is S, A, Q or W, X₄ is M, L, R or P,        X₅ is H, F, W or G, X₆ is V or A and X₇ is I or V, and of at        most the sequence SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20 or        SEQ ID No. 21 (epitope 4),    -   an epitope of at least the sequence SEQ ID No. 22, in which X₁        is Y or N, X₂ is E, D or Q, X₃ is T, N, R, M or K, X₄ is W, H or        F and X₅ is P or G, and of at most the sequence SEQ ID No. 23,        SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ        ID No. 28 or SEQ ID No. 29 (epitope 5).

According to yet another embodiment, the method of the invention uses amonoclonal antibody specific for an epitope 1 and a monoclonal antibodyspecific for an epitope 2, 3, 4 or 5. Preferably, the method of theinvention uses a monoclonal antibody specific for an epitope 1 and amonoclonal antibody specific for an epitope 2 or 4. More preferably, themethod of the invention uses a monoclonal antibody specific for anepitope 1 and a monoclonal antibody specific for an epitope 2.

The term “epitope” is intended to mean a peptide having at least thesequences as defined by the sequences SEQ ID No.s 1 to 29, and at most10, 8, 6 or 4 additional amino acids distributed on either side of thesequence under consideration, in a homogeneous or nonhomogeneous manner,or else on just one side, and also the analogues, homologues andstructural equivalents thereof.

Generally, the term “analogue” refers to peptides having a sequence anda native polypeptide structure exhibiting one or more amino acidadditions, substitutions (generally conservative in terms of nature)and/or deletions, relative to the native molecule, insofar as themodifications do not destroy the antigenic reactivity.

The analogues that are particularly preferred include substitutions thatare conservative in nature, i.e. substitutions which take place in anamino acid family. Specifically, amino acids are generally divided upinto four families, namely (1) acidic amino acids such as aspartate andglutamate, (2) basic amino acids such as lysine, arginine and histidine,(3) nonpolar amino acids such as alanine, leucine, isoleucine, proline,phenylalanine, methionine and tryptophan, and (4) uncharged polar aminoacids such as glycine, asparagine, glutamine, cysteine, serine,threonine and tyrosine. Phenylalanine, tryptophan and tyrosine aresometimes classified as aromatic amino acids. For example, it can bereasonably predicted that an isolated replacement of leucine withisoleucine or valine, of an aspartate with a glutamate, or of athreonine with a serine, or a similar conservative replacement of oneamino acid with another amino acid that is structurally related, willhave no major effect on the biological activity. Those skilled in theart will readily determine the regions of the peptide molecule ofinterest that can tolerate a change with reference to Hopp/Woods andKyte-Doolite plots, well known in the art.

The term “homology” is intended to mean the percentage identity betweentwo peptide molecules. Two amino acid sequences are “substantiallyhomologous” to one another when the sequences exhibit at least 60%,preferably at least 75%, more preferably at least 80-85%, morepreferably at least 90% and more preferably at least 95-98% or moresequence identity over a defined length of the peptide molecules.

The term “structural equivalent” is intended to mean any linear ornonlinear peptide sequence included in the protein of interest, havingthe same three-dimensional structure as the conformational epitope ofinterest, such as the epitopes of sequences SEQ ID No.s 7 to 29, whileat the same time retaining the antigenic reactivity. Such a “structuralequivalent” can be readily obtained by those skilled in the art from theconformational epitope of interest, using a bioinformatic system whichmakes it possible to find 3D structural or substructural similarities inproteins, such as the SuMo⁶⁷ or Superimposë⁶⁸ systems.

The polyclonal antibodies can be obtained by immunization of an animalwith the tumour marker concerned, followed by recovery of the desiredantibodies in purified form, by taking serum from said animal, andseparation of said antibodies from the other serum constituents, inparticular by affinity chromatography on a column to which an antigenspecifically recognized by the antibodies, in particular said marker, isattached.

The monoclonal antibodies can be obtained by the hydridoma technique,the general principle of which is summarized hereinafter.

Firstly, an animal, generally a mouse, is immunized with the tumourmarker of interest, the B lymphocytes of said animal then being capableof producing antibodies against said antigen. These antibody-producinglymphocytes are then fused with “immortal” myeloma cells (murine in theexample) so as to produce hybridomas. Using the heterogeneous mixture ofcells thus obtained, a selection of cells capable of producing aparticular antibody and of multiplying indefinitely is then carried out.Each hybridoma is multiplied in the form of a clone, each resulting inthe production of a monoclonal antibody of which the properties ofrecognition with respect to said tumour marker may be tested, forexample, by ELISA, by one-dimensional or two-dimensional Westernblotting, by immunofluorescence, or by means of a biosensor. Themonoclonal antibodies thus selected are subsequently purified, inparticular according to the affinity chromatography technique describedabove.

The monoclonal antibodies may also be recombinant antibodies obtained bygenetic engineering, by means of techniques well known to those skilledin the art.

Examples of anti-defensin-A6 molecules are known and are available inparticular in the Alpha Diagnostic International Inc. catalogue, rabbitanti-defensin-A6 polyclonal antibody, Cat. No. HDEFA61-A. No monoclonalantibody directed against Prodefensin-A6 is available to date.

Examples of anti-leukocyte elastase inhibitor antibodies are known andare available in particular in the Abcam catalogue, rabbit anti-LEIpolyclonal antibody, Cat. No. Ab47731. An anti-LEI monoclonal antibody,clone ELA-1, has been described in the article by Yasumatsu et al.⁶⁹.

Examples of anti-ezrin antibodies are known and are available inparticular in the Abeam catalogue, anti-ezrin monoclonal antibody, clone3C12, Cat. No, Ab4069 and rabbit anti-ezrin polyclonal antibody, Cat.No. Ab47418.

Examples of anti-aminoacylase 1 antibodies are known and are availablein particular in the Abnova catalogue, anti-aminoacylase 1 monoclonalantibody, clone 4F1-B7, Cat. No. H00000095-M01, and in the Abcamcatalogue, chicken anti-aminoacylase 1 polyclonal antibody, Cat. No.Ab26173.

Examples of anti-liver fatty acid-binding protein antibodies are knownand are available in particular in the Abcam catalogue, anti-L-FABPmonoclonal antibody, clone 6B6, Cat. No. Ab10059, and rabbit anti-L-FABPpolyclonal antibody, Cat. No. Ab7807.

Examples of anti-intestinal fatty acid-binding protein antibodies areknown and are available in particular in the R&D Systems catalogue,anti-I-FABP monoclonal antibody, clone 323701, Cat. No. MAB3078, and inthe Abeam catalogue, rabbit anti-I-FABP polyclonal antibody, Cat. No.Ab7805.

Examples of anti-apolipoprotein AI antibodies are known and areavailable in particular in the Biodesign Meridian Life Sciencescatalogue, anti-Apo AI monoclonal antibody, clone 4A90, Cat. No. H45402Mand goat anti-Apo AI polyclonal antibody, Cat. No. K45252P.

Examples of anti-apolipoprotein AII antibodies are known and areavailable in particular in the US Biological catalogue, anti-Apo AIImonoclonal antibody, clone 1402, Cat. No. A2299-31C and in the BiodesignMeridian Life Sciences catalogue, goat anti-Apo AII polyclonal antibody,Cat. No. K74001P.

Examples of anti-I-plastin polyclonal antibodies are known and areavailable in particular in the Santa Cruz Biotechnology catalogue. Therabbit polyclonal antibody H-300 (Cat. No. sc-28531) reacts withI-plastin, L-plastin and T-plastin. The applicant has developedmonoclonal antibodies directed against I-plastin.

Examples of anti-beta2-microglobulin, anti-CEA, anti-CA19-9 andanti-testosterone antibodies are known and are in particular used in theapplicant's assay kits, respectively Vidas® β2-Microglobulin, Vidas®CEA, Vidas® CA19-9™ and Vidas® Testosterone.

Examples of anti-proteasome 20S antibodies are known and are availablein particular in the Affinity Research Products catalogue.

Examples of anti-galectin-3, anti-L-lactate dehydrogenase chain B,anti-calreticulin, anti-tumour-associated calcium signal transducer 1,anti-keratin type II cytoskeletal 8, anti-keratin type I cytoskeletal18, anti-keratin type I cytoskeletal 19, anti-epithelial-cadherin,anti-villin and anti-TIMP-1 antibodies are known and are available inparticular in the Abcam catalogue.

Examples of anti-regenerating islet-derived protein 3 alpha antibodiesare known and are in particular used in the Dynabio assay kits (LaGaude, France).

Examples of anti-CA 242, anti-CA 50 and anti-CA 72-4 antibodies areknown and are available in particular in the Fujirebio catalogue.

Examples of anti-intelectin-1 antibody are known and are available inparticular in the Alexis Biochemicals catalogue, anti-intelectin-1monoclonal antibody, clone Saly-1, Cat. No. ALX-804-850-C100 and rabbitanti-intelectin-1 polyclonal antibody, Cat. No. ALX-210-941.

Examples of anti-cytokeratin 20 antibodies are known and are availablein particular in the Abeam catalogue, anti-cytokeratin 20 monoclonalantibody, clone Ks20.8, Cat. No. Ab962 and rabbit anti-cytokeratin 20polyclonal antibody, Cat. No. Ab36756.

Examples of anti-TCTP antibodies are known and are available inparticular in the Abnova catalogue, anti-TCTP monoclonal antibody, clone3C7, Cat. No. 157H00007178-M01 and anti-TCTP polyclonal antibody, Cat.No. 157H00007178-A01.

Examples of anti-defensin-A5 antibodies are known and are available inparticular in the Santa Cruz Biotechnology catalogue, anti-defensin-A5monoclonal antibody, clone 8C8, Cat. No. sc-53997, and in the AlphaDiagnostic International Inc. catalogue, rabbit anti-defensin-A5polyclonal antibody, Cat. No. HDEFA51-A.

The binding partners which are specific or not specific for the tumourmarker(s) sought in the method of the invention may be used as a capturereagent, as a detection reagent or as capture and detection reagents.

According to one embodiment, the binding partner specific forProdefensin-A6 is used for the capture of Prodefensin-A6, which makes itpossible to improve the specificity of the diagnostic method of theinvention.

Preferably, the monoclonal antibody which specifically recognizes anepitope 1 is used in capture and/or the monoclonal antibody whichspecifically recognizes an epitope 2, 3, 4 or 5, preferably 2, is usedin detection.

The visualization of the immunological reactions, i.e. of the tumourmarker/binding partner binding, may be carried out by any means ofdetection, such as direct or indirect means.

In the case of direct detection, i.e. without the involvement of alabel, the immunological reactions are observed, for example, by surfaceplasmon resonance or by cyclic voltametry on an electrode bearing aconductive polymer.

The indirect detection is carried out by means of labelling, either ofthe “revealing reagent” binding partner, or of the tumour marker ofinterest itself. In the latter case, this is then described as acompetition method.

The term “labelling” is intended to mean the attachment of a labelreagent capable of directly or indirectly generating a detectablesignal. A nonlimiting list of these label reagents comprises:

-   -   enzymes which produce a signal that can be detected, for        example, by colorimetry, fluorescence or luminescence, such as        horseradish peroxydase, alkaline phosphatase, β-galactosidase or        glucose-6-phosphate dehydrogenase,    -   chromophores such as fluorescent or luminescent compounds or        dyes,    -   radioactive molecules such as ³²P, ³⁵S or ¹²⁵I, and    -   fluorescent molecules such as Alexa or phycocyanins.

Indirect detection systems may also be used, such as, for example,ligands capable of reacting with an antiligand. Ligand/antiligand pairsare well known to those skilled in the art, this being the case, forexample, of the following pairs: biotin/streptavidin, hapten/antibody,antigen/antibody, peptide/antibody, sugar/lectin,polynucleotide/sequence complementary to the polynucleotide. In thiscase, it is the ligand which carries the binding partner. The antiligandmay be directly detectable by means of the label reagents described inthe previous paragraph, or may itself be detectable by means of aligand/antiligand.

These indirect detection systems may result, under certain conditions,in an amplification of the signal. This signal amplification techniqueis well known to those skilled in the art, and reference may be made tothe prior patent applications FR98/10084 or WO-A-95/08000 by theapplicant or to the article by Chevalier et al.⁷⁰.

Depending on the type of labelling used, those skilled in the art willadd reagents that make it possible to visualize the labelling.

By way of example of immunoassays as defined above, mention may be madeof “sandwich” methods such as ELISA, IRMA and RIA, “competition” methodsand direct immunodetection methods such as immunohistochemistry,immunocytochemistry, Western blotting and Dot blotting.

When the binding partner specific for Prodefensin-A6 is used in capturein a “sandwich” assay, for example the antibody specific for epitope 1,either a binding partner specific for the mature part of Prodefensin-A6(amino acids 66 to 100), or a binding partner which recognizes anepitope of the propeptide part of Prodefensin-A6 (amino acids 20-65),other than that recognized by the binding partner used for the capture,will be used in detection.

Mass spectrometry can also be used for detecting, in the biologicalfluid, the tumour marker(s) sought in the method of the invention. Theprinciple of spectrometry is widely known to those skilled in the artand is described, for example, in Patterson⁷¹.

To do this, the biological sample, which may or may not have beenpretreated, is analysed in a mass spectrometer and the spectrum obtainedis compared with that of the tumour marker(s) sought in the method ofthe invention. An example of pretreatment of the sample consists inpassing it over an immunocapture support comprising one of the bindingpartners for the tumour marker(s) sought in the method of the invention,for example an antibody directed against the tumour marker(s) sought inthe method of the invention. Another example of pretreatment of thesample may be prefractionation of the biological sample in order toseparate the proteins of the sample from one another. In techniques wellknown to those skilled in the art, the predominant proteins of thesample may, for example, first of all be depleted.

The determination of the presence, in the biological sample, of the“mRNA” tumour marker of interest may be carried out by any method fordetermining the presence of mRNA in a sample, namely either directdetection of the mRNA, or indirect detection of the mRNA, or any othermethod for determining the presence of an RNA in a sample, known tothose skilled in the art.

The term “direct detection of the mRNA” is intended to mean thedemonstration of the mRNA itself in the biological sample.

The direct detection of the mRNA in the biological sample may be carriedout by any means known to those skilled in the art, such as, forexample, by hybridization with a binding partner specific for the mRNA,where appropriate after amplification by the PCR or NASBA technique.

The term “hybridization” is intended to mean the process during which,under suitable conditions, two nucleotide fragments bind to one anotherwith stable and specific hydrogen bonds so as to form a double-strandedcomplex. These hydrogen bonds form between the complementary basesadenine (A) and thymine (T) (or uracil (U)) (referred to as an A-T bond)or between the complementary bases guanine (G) and cytosine (C)(referred to as a G-C bond). The hybridization of two nucleotidefragments may be complete (reference is then made to complementarynucleotide fragments or sequences), i.e. the double-stranded complexobtained during this hybridization comprises only A-T bonds and C-Gbonds. This hybridization may be partial (reference is then made tosufficiently complementary nucleotide fragments or sequences), i.e. thedouble-stranded complex obtained comprises A-T bonds and C-G bonds whichmake it possible to form the double-stranded complex, but also basesthat are not bonded to a complementary base. The hybridization betweentwo nucleotide fragments depends on the operating conditions that areused, and in particular the stringency. The stringency is defined inparticular as a function of the base composition of the two nucleotidefragments, and also by the degree of mismatching between two nucleotidefragments. The stringency can also depend on the reaction parameters,such as the concentration and the type of ionic species present in thehybridization solution, the nature and the concentration of denaturingagents and/or the hybridization temperature. All these data are wellknown and the appropriate conditions can be determined by those skilledin the art. In general, depending on the length of the nucleotidefragments that it is desired to hybridize, the hybridization temperatureis between approximately 20 and 70° C., in particular between 35 and 65°C., in a saline solution at a concentration of approximately 0.5 to 1 M.The binding partners which are specific or not specific for the mRNA areany partner capable of binding to this mRNA. By way of example, mentionmay be made of nucleic probes, amplification primers, and any othermolecule capable of binding to this mRNA.

The term “hybridization probe” is intended to mean a nucleotide fragmentcomprising from 5 to 100 nucleic units, in particular from 10 to 35nucleic units, having a hybridization specificity under given conditionsso as to form a hybridization complex with the material specific for thetarget gene of interest. The hybridization probe may comprise a labelenabling its detection.

For the purpose of the present invention, the term “amplificationprimer” is intended to mean a nucleotide fragment comprising from 5 to100 nucleic units, preferably from 15 to 30 nucleic units, enabling theinitiation of an enzymatic polymerization, such as, in particular, anenzymatic amplification reaction. The term “enzymatic amplificationreaction” is intended to mean a process that generates multiple copiesof a nucleotide fragment via the action of at least one enzyme. Suchamplification reactions are well known to those skilled in the art andmention may in particular be made of the following techniques:

-   -   PCR (polymerase chain reaction), as described in U.S. Pat. No.        4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159,    -   NASBA (nucleic acid sequence-based amplification) with patent        application WO 91/02818, and    -   TMA (transcription mediated amplification) with U.S. Pat. No.        5,399,491.

The term “detection” is intended to mean either a physical method, or achemical method with an intercalating dye such as SYBR® Green I orethidium bromide, or a method of detection using a label. Many detectionmethods exist for detecting nucleic acids⁷². The appropriate labels areas defined above.

For the purpose of the present invention, the hybridization probe may bea “detection” probe. In this case, the “detection” probe is labelled bymeans of a label as defined above. By virtue of the presence of thislabel, it is possible to detect the presence of a hybridization reactionbetween a given detection probe and the transcript to be detected.

The detection probe may in particular be a “molecular beacon” detectionprobe⁷³. These “molecular beacons” become fluorescent duringhybridization. They have a stem-loop structure and contain a fluorophoreand a quencher group. The binding of the specific loop sequence with itscomplementary target nucleic acid sequence causes unfolding of the stemand the emission of a fluorescent signal during excitation at theappropriate wavelength.

The hybridization probe may also be a “capture” probe. In this case, the“capture” probe is immobilized or can be immobilized on a solid supportby any appropriate means, i.e. directly or indirectly, for example bycovalence or adsorption. The appropriate solid supports are known tothose skilled in the art, and, by way of examples, mention may be madeof synthetic materials or natural materials, latices, magneticparticles, metal derivatives, gels, etc. The solid support may be in theform of a microtitration plate, a membrane as described in applicationWO-A-94/12670 or a particle. It is also possible to immobilize severaldifferent capture probes on the support, each capture probe beingspecific for a target transcript. In particular, it is possible to use,as support, a biochip on which a large number of probes may beimmobilized.

The immobilization of the probes on the support is also known to thoseskilled in the art, and mention may be made of a deposit of probes bydirect transfer, microdeposition, in situ synthesis andphotolithography.

The demonstration, in the biological sample, of the DNA modifications oranomalies in the gene encoding the tumour marker of interest may becarried out by any method for determining DNA alterations in a sample,namely either the direct detection of mutations, or the demonstration ofalterations in the methylation profile of the loci of interest, or anyother method for determining DNA alterations in a sample, known to thoseskilled in the art.

The mutations may include point substitutions of one nucleotide withanother, deletions of one or more nucleotides and insertions of one ormore nucleotides. The mutations may be located in the coding portion ofthe gene of the tumour marker of interest, or in the 5′ and 3′ noncodingportions, such as the transcription promoter region or the transcriptiontermination region.

The strategies for demonstrating a mutation are based on molecularbiology techniques and comprise steps of DNA extraction, amplificationby PCR or another amplification technique, hybridization and/orsequencing. In the case of colorectal cancer, the following method hasbeen successfully used to detect mutations in faecal DNA: concentrationof the DNA by precipitation, enrichment in the target using captureoligonucleotides on magnetic beads, PCR amplification of the genes ofinterest, solid-phase sequencing for identifying point mutations⁷⁴. Thedeletions were identified with respect to the difference in size betweenthe expected reference fragment and the mutated fragment. Imperiale etal.⁷⁴ have described a panel of 21 mutations located in the K-ras, APCand p53 genes, which makes it possible to detect 16/31 of invasivecancers.

Other DNA markers used are the BAT-26 deletion, which is a marker forinstability of microsatellites and highly amplifiable DNA called longDNA (L-DNA), which is not a specific marker but which appears to reflectthe disorganized apoptosis of exfoliated tumour cells in the coloniclumen⁷⁵. These markers are not satisfactory, either in terms of theirsensitivity or in terms of their specificity.

As previously indicated, the DNA alterations may also correspond to amodification of the methylation profile of the gene corresponding to thetumour marker of interest. The modification of the methylation profilemay correspond to a hypomethylation (decrease in the number ofmethylations) or to a hypermethylation (increase in the number ofmethylations). The altered units may be located in the coding portion ofthe gene of the tumour marker of interest, or in the 5′ and 3′ noncodingportions, such as the transcription promoter region or the transcriptiontermination region.

The analysis of the DNA methylation may be carried out using techniquesbased on qualitative and/or quantitative PCR, such as MSP(methylation-specific PCR), bisulphite sequencing, digestion with amethylation-sensitive restriction enzyme coupled with PCR, COBRA(combined bisulphite restriction analysis) and Ms-SNuPE(methylation-sensitive single nucleotide primer extension). All thesetechniques have been reviewed comparatively and in detail in amethodology article⁷⁶.

In the literature, several hypermethylated genes have been reported inthe case of colorectal cancer. By way of example, mention may be made ofthe ALX4 (aristaless-like homeobox-4) gene⁶⁰, the promoter region of theTPEF/HHP1 (transmembrane protein containing epidermal growth factor andfollistatin domain) gene⁷⁷ or else the septin-9 gene⁷⁸.

When, in the method of the invention, at least two markers are detected,they may be demonstrated separately, for example by means of differentimmunoassay determinations, or else simultaneously, in a multiplexassay.

When, in the method of the invention, two markers of different natureare detected, for example a protein marker and an mRNA marker, twodifferent detection methods, chosen from those described above, may beused. They may also be detected simultaneously, in the same detectionmedium and under the same reaction conditions, as described in patentapplication WO 03/104490. The steps of the detection method described inthis patent application, which consists in simultaneously detectinghybridization and immunological reactions in a sample that may containtarget analytes constituted of at least one nucleic acid and of at leastone other ligand of different nature, consist in:

(i) depositing a known volume amount of the sample diluted in a reactionbuffer, on a capture surface precoated with capture partners for saidtarget analytes, said capture partners comprising at least one nucleicprobe and at least one antiligand,

(ii) reacting at a temperature of between 15° C. and 60° C., and

(iii) visualizing the hybridization and immunological reactions thusobtained.

The biological sample may require a particular treatment because it maycontain the tumour marker(s) sought in the method of the invention, assuch, or else it may contain circulating tumour cells which contain themarkers sought in the method of the invention and/or circulating tumourcells which are capable of secreting the marker(s) sought in the methodof the invention.

Thus, according to one embodiment of the invention, the biologicalsample is pretreated in order to isolate the circulating tumour cellscontained in said fluid.

The expression “isolate circulating tumour cells” is intended to meanobtain a cell fraction enriched in circulating tumour cells.

The treatment of the biological sample in order to isolate thecirculating tumour cells can be carried out by cell sorting in a flowcytometer, by enrichment on Ficoll, by enrichment with magnetic beadscovered with specific antibodies, or by any other method of specificenrichment known to those skilled in the art.

In the case of blood as biological sample, the circulating tumour cellsmay be isolated by means of a technique of cell separation on Ficollcombined with depletion of the blood cells using anti-CD45 antibodiescoupled to magnetic beads (Dynal Biotech ASA, Norway).

The detection of the tumour marker(s) sought in the method of theinvention can then be carried out directly using circulating tumourcells isolated from the biological sample, for example byimmunocytochemical labelling of these cells with an antibody againsttumour marker(s) sought in the method of the invention, after havingdeposited the circulating tumour cells on a slide by cytospin. Thedetection of the tumour marker(s) sought in the method of the inventionmay also be carried out directly in the circulating tumour cells usingthe flow cytometry method as described in Métézeau et al.⁷⁹.

Under these conditions, said circulating cells can be treated underconditions which make it possible to block the tumour marker(s) soughtin the method of the invention, inside said cells. Such a treatment isdescribed by Mathieu et al.⁸⁰.

The detection of the tumour marker(s) sought in the method of theinvention is then carried out after having made the cell membranepermeable so as to allow entry of the binding partners specific for themarker(s) sought in the method of the invention.

The direct detection of the tumour marker(s) used in the method of theinvention, based on the circulating cells, may also be carried out bymeans of an ELISPOT method, for example by means of the method describedin patent application WO 03/076942 filed by the applicant. This methodis a method for detecting and/or quantifying circulating tumour cells ofa biological sample, which are capable of releasing or secreting, invitro, one or more tumour marker(s), comprising the steps consisting in:

(i) depositing an amount of said cells at the bottom of a culturesurface to which at least one binding partner specific for said tumourmarker(s) is attached,

(ii) culturing said cells under conditions such that they release orsecrete said tumour markers, which are immunocaptured at the bottom ofthe culture surface,

(iii) removing the cells by washing,

(iv) adding at least one labelled conjugate specific for said tumourmarkers, and

(v) visualizing the labelling thus obtained.

The direct detection of the tumour marker(s) used in the method of theinvention in the tumour cells may also be carried out in the culturemedium of said cells after having cultured them under conditions suchthat they secrete tumour marker(s) used in the method of the invention.

The culture conditions for release or the expression of the tumourmarkers are conventional conditions such as 37° C. under a humidatmosphere and at 5% CO₂.

When the biological sample is a solid sample, the presence of the tumourmarker(s) may also be shown in vivo, in situ in the tumours.

In order to show the presence of a tumour marker in a tumour in vivo,any imaging method known to those skilled in the art may be used. Forthis, a binding partner for said tumour marker may be coupled to animaging tracer.

The term “coupling of the binding partners to an imaging tracer” isintended to mean the attachment of a tracer capable of being detected byany imaging method known to those skilled in the art, and of directly orindirectly generating a detectable signal. Thus, the tracer may be aradioactive tracer such as technetium-99. In this case, the organ whichhas the primary cancer or the metastases will bind the tumour marker andits tracer. The radiation emitted by the organ can be filmed with aspecial camera, for example a gamma-camera. The instrument collects thescintillations generated by the radioactive substance and thus makes itpossible to visualize the organ.

In another method of the invention, the tracer may comprise apositron-emitting radioactive substance (fluorine 18). The images willthen be acquired by a positron emission tomography system.

In another preferred method of the invention, the partner of the tumourmarker(s) may be coupled to nanoparticles. By way of example, they maybe supramagnetic nanoparticles; for example, anionic magneticnanoparticles for use in direct cell labelling and in vivo detection bynuclear magnetic resonance imaging. They may also be gold nanoparticles.

By virtue of the methods of the invention which make it possible todetect the tumour marker in vivo, it will be possible to visualize theareas in the body where there has been binding of the tumour markerbinding partner, the cancers producing the tumour marker, and inparticular colorectal cancer, and also the locations of their remotemetastases and the lymph node involvement.

The method of the invention can be used both for early diagnosis, andfor screening, therapeutic follow-up, prognosis and relapse diagnosis inrelation to colorectal cancer since only the cancer cells secreteProdefensin-A6 and this production depends on the grade of the cancer,which constitutes another subject of the invention.

The invention will be understood more clearly by means of the followingexamples given by way of nonlimiting illustration, and also by means ofthe appended FIGS. 1 to 6, in which:

FIG. 1 represents the comparison, by the Western blotting technique, ofthe nine anti-Prodefensin-A6 monoclonal antibodies. The graphs show thesignals corresponding to the volume of the band (in intensity*mm²) forthe Prodefensin-A6 peptide (SEQ ID No. 1, 7 ng/well) (FIG. 1A) and forthe Prodefensin-A6 protein secreted into the transfected 293T culturesupernatants (FIG. 1B). The exact values of the band volumes areindicated in the table (FIG. 1C);

FIG. 2 represents the comparison, by the dot-blotting technique, of thenine anti-Prodefensin-A6 monoclonal antibodies. The graphs show thesignals corresponding to the average volume of two spots (inintensity*mm²) for the Prodefensin-A6 peptide (SEQ ID No. 1) (FIG. 2A)and for the Prodefensin-A6 propeptide (SEQ ID No. 3) (FIG. 2B). Theexact values of the band volumes are indicated in the table (FIG. 2C).The ratio*100 given in this table is calculated according to theformula: Prodefensin-A6 propeptide signal/Prodefensin-A6 peptidesignal*100;

FIG. 3 is a graph relating to the analysis of the recognition of theProdefensin-A6 propeptide by the indirect ELISA technique. The exactvalues of the ELISA signal in absorbance units are indicated in thetable next to the graph;

FIG. 4 recapitulates the sequences recognized by each of theanti-Prodefensin-A6 antibodies 1H8C9, 11B2D2, 11E8C9 and 13E7F3. Theamino acid sequence of each motif displayed by a phage library andbinding to the antibody studied is given in the “immunoreactive motifs”box. For each antibody, these immunoreactive motifs are aligned in orderto determine the consensus sequence which is indicated in bold;

FIG. 5 represents the analysis of the reactivity by sandwich ELISA ofthe five anti-Prodefensin-A6 monoclonal antibodies directed againstepitope 1 with the 1H8C9 detection antibody. The antigen used is thetransfected 293T culture supernatant containing secreted Prodefensin-A6.This supernatant was diluted to 1/100 or 1/200. The exact values of theELISA signal in RFV are indicated in the table next to the graph;

FIG. 6 is a graph relating to the assaying by ELISA of Prodefensin-A6,in pg/ml, in the serum of patients having a colorectal adenocarcinoma(CRC+), of healthy individuals (CRC−), of patients having aninflammatory digestive disease (IDD) and of patients having a colorectaladenoma;

FIG. 7 represents the optimization of the SPE fractionation on MCXcartridges for the peptide EPLQAEDDPLQAK (SEQ ID No. 30) ofProdefensin-A6. The graphs represent the area of the peak correspondingto transition 727/556 of the peptide as a function of the pH of thebuffer used to carry out the fractionation on an MCX cartridge. FIG. 7A:Prodefensin-A6 dissolved in water; FIG. 7B: Prodefensin-A6 dissolved inhuman serum from healthy individuals.

EXAMPLE 1 Cloning of the Genes Encoding the Tumour Markers andExpression of Recombinant Proteins

For the aminoacylase-1, LEI, L-FABP, ezrin, I-plastin, Gal-3, villin,I-FABP and calreticulin tumour markers, the cDNA amplification andcloning, the construction of the expression vectors and also theexpression and purification of the recombinant proteins were describedin detail in patent application WO2009/024691.

1. Expression of the Prodefensin-A6 Protein in Human Cells byTransfection

An expression vector containing the cDNA of the Prodefensin-A6 gene(pCMV6-XL5 DEFA6) was purchased from the company Origene (Cat, No.SC303095). This vector makes it possible to express the Prodefensin-A6protein under the control of the CMV promoter after introduction intomammanian cells.

HEK 293T human embryonic kidney cells were maintained in culture in DMEMmedium containing 10% FCS, at 37° C. with 5% CO₂. The cell layersbetween 50% and 70% confluence were transfected with the pCMV6-XL5 DEFA6expression plasmid using one of the following two transfection reagents:Lipofectamine LTX sold by Invitrogen (Cat No. 15338-100) or TransIT LT-1(Cat No, MIR2300) sold by Euromedex. In both cases, the transfectionswere carried out according to the procedure provided by the producers ofthe reagent. After transfection, the cultures were incubated for 48 h inorder to allow the production of the Prodefensin-A6 protein. Next, theculture supernatant was harvested, centrifuged, and then filtered so asto remove cell debris. The Prodefensin protein secreted into the culturesupernatant underwent all the post-translational modifications necessaryfor its folding; it is native. It is this supernatant that was used as asource of Prodefensin-A6 protein in the screening of theanti-Prodefensin-A6 monoclonal antibodies and in the characterization ofthese antibodies.

2. Chemical Synthesis of Peptides

Three peptides corresponding to various parts of the Prodefensin-A6protein were obtained by chemical synthesis according to procedures wellknown to those skilled in the art (NeoMPS). The peptide of which thesequence is indicated in SEQ ID No. 1 corresponds to the entire sequenceof Prodefensin-A6 without the signal peptide which is cleaved during thetranslocation of the polypeptide chain to the endoplasmic reticulum.This peptide is called Prodefensin-A6 peptide or Prodefensin-A6precursor. The peptide of which the sequence is indicated in SEQ ID No.2 corresponds to the whole of the propeptide part of the Prodefensin-A6precursor. This peptide is called Prodefensin-A6 propeptide orpropeptide part of Prodefensin-A6. The peptide of which the sequence isindicated in SEQ ID No. 3 corresponds to virtually the whole of thepropepide part of the Prodefensin-A6 precursor. Four amino acids of theN-terminal end are not included in the sequence. This peptide is easierto produce in large amounts than the peptide of sequence SED ID No. 2and was used as a replacement when the presence of the four amino acidsis not essential.

SEQ ID No. 1: EPLQAEDDPLQAKAYEADAQEQRGANDQDFAVSFAEDASSSLRALGSTRAFTCHCRRSCYSTEYSYGTCTVMGINHRFCCL SEQ ID No. 2: EPLQAEDDPLQAKAYEADAQEQRGANDQDFAVSFAEDASSSLRALGS SEQ ID No. 3: AEDDPLQAKAYEADAQEQRGANDQDFAVSFAEDASSSLRALGS

EXAMPLE 2 Production of Monoclonal Antibodies Directed Against theTumour Markers

1. Animal Model

The immunization experiments were carried out in female BALB/c (H-2^(d))mice aged 6 to 8 weeks at the time of the first immunization.

2 Immunogens and Immunizations

In order to increase the immune responses obtained in the mice and to beable to generate monoclonal antibodies, the tumour markers were producedin the form of recombinant proteins or of synthetic peptides producedaccording to the procedures described in example 1. The LDH protein wasobtained from the company SciPac (Cat. No. 103-133). When the syntheticpeptides were used as immunogen, they were coupled to carrier proteinssuch as bovine serum albumin (BSA) or KLH (keyhole limpet haemocyanin).These proteins were mixed volume for volume with Freund's adjuvant(Sigma), prepared in the form of a water-in-oil emulsion and which isknown to have a good immunogenic capacity. Three mice were immunized foreach tumour marker. The mice received three successive doses of 10 μg ofthe immunogens at 0, 2 and 4 weeks. All the injections were givensubcutaneously. The first injection is given as a mixture of completeFreund's adjuvant, the following two are given as a mixture withincomplete Freund's adjvant. Between D50 and D70 after the firstinjection, the humoral responses were restimulated with an intravenousinjection of 100 μg of the recombinant protein. For Prodefensin-A6, twodifferent series of immunizations were carried out. A first group ofmice received the Prodefensin-A6 peptide (SEQ ID No. 1) coupled to BSAand to KLH (in alternation according to injections). A second group ofmice received the Prodefensin-A6 propeptide (SEQ ID No. 2) coupled toBSA and to KLH (in alternation according to injections).Anti-Prodefensin-A6 antibodies were obtained from the animals belongingto each of the two groups.

3. Monitoring the Appearance of the Humoral Response

In order to monitor the appearance of the antibodies, blood samples weretaken regularly from the mice. The presence of the anti-tumour markerantibodies is tested using an ELISA. The protein of interest is used forcapture (1 μg/well); after saturation, various dilutions of the testsera are reacted with the antigen (incubation at 37° C., for 1 h). Thespecific antibodies present in the serum are revealed with an AffiniPuregoat anti-mouse IgG antibody conjugated to alkaline phosphatase (H+L,Jackson Immunoresearch, Cat No. 115-055-146), which binds to theantibodies being sought (0.1 μg/well).

4. Production of Monoclonal Antibodies

Three days after the final injection, for each tumour marker, one of themice immunized was sacrificed; the blood and the spleen were removed.The splenocytes obtained from the spleen were cultured with Sp2/0-Ag14myeloma cells in order for them to fuse and become immortilized,according to the protocol described by Köhler and Milstein^(81,82).After an incubation period of 12-14 days, the supernatants of thehydridomas obtained were screened in order to determine the presence ofanti-tumour marker antibodies, using the ELISA assay described in point3 of this example. When synthetic peptides coupled to BSA or KLH wereused as immunogen, the clones directed against BSA and KLH wereeliminated by carrying out an ELISA screening with uncoupled BSA or KLHfor capture. The positive hybridoma colonies were subcloned twiceaccording to the limiting dilution technique, which is well known tothose skilled in the art.

5. Characterization of the Monoclonal Antibodies by Immunoblotting

The monoclonal antibodies directed against the aminoacylase-1, LEI,ezrin, plastin, Gal-3, calreticulin and LDH tumour markers are describedin patent application WO2009/024691.

The 1H8C9, 11B2D2, 13E7F3, 11E8C9, 6E1B4, 10C2G3, 12F3F2, 12F8E4 and12H4E1 monoclonal antibodies are directed against Prodefensin-A6 andwere obtained by carrying out the techniques described in points 1 to 4of this example.

5.1. Methodology

The transfected 293T line cell culture extracts are prepared by directlylysing the cell pellet with 600 μl of PBS containing 0.5% Triton X-100and protease inhibitors, and then treated according to the NuPAGE Novexgel sample preparation protocol (Invitrogen). To obtain the tissueextracts, tumour and mucosal biopsies of patient CLSP105 weredissociated with a scalpel, and were then subjected to ten cycles ofextraction in the Medimachine system (Becton Dickinson) using 50 μmMedicons with 1 ml of PBS buffer containing 2.5 mM EDTA and proteaseinhibitors (Roche Complete™ tablets). These 10 ml of cell suspension arepooled, made up to 25 ml, and then centrifuged for 15 min at 600 g. Thesupernatant corresponds to the tissue extract which is treated accordingto the NuPAGE Novex gel sample preparation protocol. Reduced samples areused, at a final total protein concentration of 0.4 mg/ml. The depositvolume is 20 μl per well, on a NuPAGE Novex bis-tris 4-12% gel, with MESrunning buffer. After migration (at 200 V, for 1 hour), and transferonto a PVDF membrane (at 400 mA, for 45 min), the quality of thetransfer is assessed by staining with amido black.

The membranes are saturated with 5% skimmed milk (Régilait) in asolution of TNT (15 mM Tris, 0.14 M NaCl, 0.5% Tween 20, pH 8) atambient temperature for 1 hour, After saturation, the membranes areincubated for 1 hour with the various test antibodies diluted to 10μg/ml in the saturating solution. After rinsing with TNT, the membranesare incubated for 1 hour at ambient temperature with an anti-mousehorseradish peroxidase conjugate diluted to 1:5000 (Cat No. 115-035-062,Jackson Immunoresearch) in the saturating solution. After rinsing, thedeveloping is carried out with the Substrate Supersignal West DuraExtended kit (Cat No. 34076, Pierce) according to the recommendedinformation for use. The exposure time was 2 minutes for the experimentpresented in FIGS. 1 and 100 seconds for the experiment presented inTable 1.

The chemiluminescence signal on the membranes was measured with theVersaDoc 5000 imaging system from BioRad. Based on the image of theWestern blot, the volumes of the bands which correspond to the varioustumour markers were evaluated with the QuantityOne software (Bio-Rad).The volume corresponds to the intensity of the chemiluminescence signalmultiplied by the surface area of the band.

5.2. Results

The Western blotting results reproduced in FIG. 1 and Table 1 give thevolume of the bands corresponding to the tumour marker of interest forthe Western blotting analyses, as a function of the various samplestested. The culture supernatant of 293 Ts transfected with thepCMX-XL5-Prodefensin-A6 plasmid containing secreted Prodefensin-A6 andthe Prodefensin-A6 peptide (7 ng per well) were used to evaluate thereactivity in Western blotting of the nine anti-Prodefensin-A6antibodies (FIG. 1). All the antibodies recognize the Prodefensin-A6peptide. All the antibodies, except for the monoclonal 11E8C9, recognizethe Prodefensin-A6 secreted into the culture supernatant under theexperimental conditions used. However, intensity of the signals obtainedis very disparate, showing that not all the antibodies have the samereactivity. The results presented in Table 1 show that the tumourmarkers tested are well expressed in the tumour tissue obtained from thepatients. Prodefensin-A6 is not expressed by the Caco-2 or HT-29 coloncell lines; for this reason, these lysates were not included in theanalyses.

The intensity of the signal obtained with an antibody on a sample can becompared to the signals obtained with the other samples and the sameantibody. The technique used makes it possible to confirm the presenceor the absence of the marker in the tissue (non-remote sample) and thespecificity of the antibodies with respect to the markers. Thistechnique has not been used in this example in the remote samplesbecause it would not make it possible to come to a conclusion regardingthe presence or absence of the tumour marker in the remote samples, norto determine whether the concentration of said tumour marker isincreased or decreased in said samples. Furthermore, the experimentalscheme used does not make it possible to compare the reactivity of oneantibody with another.

TABLE 1 Anti- 293T/ProDEFA6 CLSP105 CLSP105 ProDEFA6 culture mucosaltumour antibody supernatant tissue tissue 1H8C9 2475 Negative 457 11B2D2556 Negative 140 13E7F3 134 Negative Negative 11E8C9 Negative NegativeNegative

6. Analysis of the Recognition of the Prodefensin-A6 Propeptide by theMonoclonal Antibodies by Dot-Blotting

6.1. Methodology

The dot-blotting analysis was carried out using the Prodefensin-A6peptide (SEQ ID No. 1, concentration 0.1 mg/ml) and the Prodefensin-A6propeptide (SEQ ID No. 3, concentration 2.38 mg/ml). A drop of eachsample was deposited in duplicate onto nitrocellulose membranes(Transblot transfer medium, Bio-rad) and then dried in the open air.

The immunodeveloping protocol is identical to that described inparagraph 5.1 of this example. The exposure time used for thisexperiment was 1 minute.

6.2. Results

All the antibodies tested recognize the Prodefensin-A6 peptide (SEQ IDNo. 1) in dot-blotting (FIG. 2). The most reactive antibody in thistechnique is the 10C2G3 clone. The 6E1B4, 12F3F2, 12F8E4 and 12H4E1antibodies form a homogeneous group, less reactive than 10C2G3 but morereactive than the group 1H8C9, 11B2D2, 13E7F3 and 11E8C9, which is moreheterogeneous. Moreover, the reactivities in Western blotting and indot-blotting do not always correlate: the 6E1B4, 12F3F2, 12F8E4 and12H4E1 antibodies have a similar reactivity in dot-blotting, whereas theintensities of the signals obtained in Western blotting are different(FIGS. 1 and 2). The Western blotting was carried out under denaturingconditions, whereas the peptides are spotted directly withoutdenaturation for the dot-blotting.

Only the 6E1B4, 10C2G3, 12F3F2, 12F8E4 and 12H4E1 antibodies recognizethe Prodefensin-A6 propeptide (SEQ ID No. 3) in dot-blotting. The 1H8C9,11B2D2, 13E7F3 and 11E8C9 antibodies do not show any significantreactivity with the Prodefensin-A6 propeptide (SEQ ID No. 3), indicatingthat their epitope is located in the mature defensin-A6 part of theProdefensin-A6 precursor. Among the five antibodies which react with theProdefensin-A6 propeptide (SEQ ID No. 3), the minimum epitope of whichis therefore contained in this sequence, the 6E1B4, 10C2G3, 12F3F2 and12F8E4 clones all exhibit the same reactivity profile: the ratio of thesignals obtained (Prodefensin-A6 propeptide/Prodefensin-A6 peptide*100,FIG. 2) is between 29 and 31 for all these antibodies. The 12H4E1 clonediffers from these other anti-Prodefensin-A6 propeptide antibodiesbecause this ratio is 6. It recognizes the propeptide less well than theother four antibodies.

7. Analysis of the Recognition of the Prodefensin-A6 Propeptide b theMonoclonal Antibodies by Indirect ELISA

7.1. Methodology

96-Well plates (of the Nunc, Maxisorp type) were coated with theProdefensin-A6 propeptide (SEQ ID No. 3) diluted in PBS to 2 μg perwell, overnight at ambient temperature. The plates are saturated with10% milk in PBS-0.05% Tween 20 (PBS-T) for 1 h at 37° C. Three washes inPBS-T are carried out, 0.2 μg/well of the biotinylated test antibody,diluted in PBS-T containing 1% BSA, is deposited on the plates andincubation is carried out for 1 h at 37° C. After three PBS-T washes,streptavidin coupled to horseradish peroxidase (Jackson ImmunoresearchCat. No. 016-030-084, dilution 1/20 000 in PBS-T containing 1% BSA, 100μl/well) is added. After incubation for 1 h at 37° C. and three PBS-Twashes, the OPT EIA substrate (BD), 1000/well, is added. After 20 min,when the coloration develops, the reaction is stopped with 5 N sulphuricacid and the absorbance at 450 nm is measured. Results presented are theaverage of two measurements, the average background noise (0.02 opticaldensity units) has not been subtracted.

7.2. Results

The results of the indirect ELISA assay are presented in FIG. 3. Underthe experimental conditions used, only the four antibodies 6E1B4,10C2G3, 12F3F2 and 12F8E4 bind to the Prodefensin-A6 propeptide adsorbedonto the solid phase. The other five antibodies, including 12H4E1, donot bind to the propeptide in this format. This experiment confirms thedot-blotting results (FIG. 2) and definitively establishes that, eventhough the 12H4E1 monoclonal antibody recognizes the Prodefensin-A6propeptide, its reactivity is much less in comparison with the otherfour monoclonals 6E1B4, 10C2G3, 12F3F2 and 12F8E4.

8. Characterization of the Epitopes Recognized by the MonoclonalAntibodies Using the Spotscan and Phage Display Peptide LibraryScreening Techniques

8.1. Methodology

The Spotscan technique, adapted according to Frank and Döring⁷⁸, makesit possible to simultaneously synthesize a large number of peptidesbound to a cellulose membrane. These peptides reproduce the sequence ofthe target antigen in the form of peptides of 8 to 12 amino acids,overlapping by 1 to 4 residues. These peptides are then brought intocontact with the antibody to be studied in a colorimetric test of blottype, and the identification of the immunoreactive peptides makes itpossible to deduce the minimum sequence of the antibody epitope and tolocate it precisely on the antigen.

The synthesis is carried out on a cellulose membrane uniformly bearingpolyethylene glycol (PEG) arms of 8 to 10 units in length, having a freeNH₂ function at the chain end. It takes place from the C-terminal end tothe N-terminal end of the peptides. The amino function of the aminoacids is detected with an Fmoc (9-fluoromethyloxycarbonyl) group, andtheir side chains, capable of reacting during the synthesis, are alsoprotected with trityl, t-butyl or t-butyl ether groups. The stocksolutions of amino acids are prepared at a concentration of 0.33 M inMNP (N-methylpyrrolidone) containing 0.5 M of HOBt(hydroxybenzotriazole). The amino acids are deposited using the ASP 222robot (Abimed, Langenfeld, Germany), controlled by means of the AutoSpotXL software. The use of this robot makes it possible to simultaneouslyproduce up to four membranes of 96 spots. i.e. 384 peptides.

For one amino acid coupling cycle, the robot deposits 0.7 μl of thesolution of extemporaneously activated amino acid (one volume ofsolution of 1.1 M diisopropylcarbodiimide diluted in NMP for threevolumes of amino acid stock solution) onto the membranes. Thisdepositing is repeated a second time, and then the membranes are rinsedin DMF (N,N-dimethylformamide). The NH₂ groups which have not reactedare then acetylated via 4 to 6 incubations for 10 minutes in a 10%solution of acetic anhydride in DMF, in order to prevent the appearanceof abortive or truncated peptides. After three washes of 2 minutes inDMF, the Fmoc groups protecting the amino function of the amino acidsare cleaved by incubation for 5 minutes in a 20% piperidine solution inDMF. After four washes in DMF, the spots are coloured using a 1%solution of bromophenol blue in DMF, and then the membrane is rinsedthree times in methanol and dried in the open air before the subsequentcoupling cycle.

This protocol is repeated for the addition of each new amino acid. Afterthe coupling of the final amino acid, the peptides are acetylated inorder to enable the blocking of all the free NH₂ groups, thus preventingthe addition of another amino acid. The side chains of all the peptidesare then deprotected by incubating the membranes in a trifluoroaceticacid/dichloromethane/triisobutylsilane (5:5:0.3) bath for 1 hour. Themembranes are then rinsed four times in dichloromethane, three times inDMF and three times in methanol, before being dried in the open air andstored at −20° C. until the immunodeveloping.

In order to immunodevelop the spots with a monoclonal antibody, themembranes are first rinsed in methanol, and then washed in TBS (50 mMTris-HCl, pH 8.0, 140 mM NaCl, 3 mM KCl), before being incubatedovernight at ambient temperature in the saturating solution(casein-based 10× concentrated solution (Western Blocking reagent,Roche) diluted in TBS-0.05% Tween 20 (TBS-T) and containing 5% sucrose).After a wash for 10 minutes in TBS-T, the membranes are incubated for 1h 30 min at 37° C. with the monoclonal antibody diluted to 20 μg/ml insaturating solution. The membranes are then washed three times in TBS-T,and then incubated with the alkaline phosphatase-coupled anti-mouseconjugate (Jackson Immunoresearch), diluted to 1/2000th in saturatingsolution. After two washes for 10 minutes in TBS-T, and then two washesin CBS (10 mM citric acid, pH 7, 140 mM NaCl, 3 mM KCl), the developer,prepared extemporaneously (600 μM 5-bromo-4-chloro-3-indoyl phosphate,720 μM thiazolyl blue tetrazolium bromide and 5 mM MgCl₂ in CBS), isbrought into contact with the membrane for 30 to 45 minutes in the dark.The immunoreactive peptides appear in blue-violet. After three rinses indistilled water, the membranes are scanned and then stored in wateruntil regeneration.

The regeneration makes it possible to remove the antibodies and theconjugates bound to the peptides, which thus makes it possible to carryout a further immunoreactivity test with respect to another antibody.The membranes undergo a series of washes, each of 10 minutes: one washin distilled water, six washes in DMF, three washes in regeneratingbuffer A (8 M urea, 35 mM SDS (sodium dodecyl sulphate), 0.1%β-mercaptoethanol), three washes in regenerating buffer B (distilledwater/ethanol/acetic acid 4:5:1), and then two washes in methanol. Themembranes are then dried in the open air before being stored at −20° C.

The characterization of the epitopes by screening of phage displaypeptide libraries was carried out using the commercial PhD12 PhageDisplay Peptide Library Kit (Cat. No. E#8110S) from New England Biolabs,according to the instructions supplied with the kit, version 2.7 of theprotocol dated November 2007.

8.2. Results

Table 2 reproduces the epitopes recognized by five anti-Prodefensin-A6propeptide antibodies, the epitope of which was analysed by the Spotscantechnique. All these antibodies recognize the same region of theProdefensin-A6 precursor, located between amino acids 25 and 30according to the numbering beginning at the initial methionine (epitope1). On the other hand, the exact sequence recognized by the variousmonoclonals is not identical. Thus, the 10C2G3 clone recognizes aminimum sequence of two amino acids (DP), whereas the 12H4E1 and 6E1B4clones require a minimum sequence of six amino acids (EDDPLQ) in orderto bind. The results presented in FIGS. 2 and 3 show that it is the10C2G3 clone which makes it possible to obtain the highest signal in dotblotting and indirect ELISA for the recognition of the Prodefensin-A6peptides. Surprisingly, the 12H4E1 clone differs from the otherantibodies directed against epitope 1 of Prodefensin-A6 (FIGS. 2 and 3),even though the minimum sequence recognized is identical with the 6E1B4antibody. The 12H4E1 clone recognizes the EDDPLQ sequence (SEQ ID No. 6)better in the context of the total precursor protein (SEQ ID No. 1) thanin the context of the Prodefensin-A6 propeptide (SEQ ID No. 3).

TABLE 2 Epitope Sequence of the epitope^(a) Antibodies No. (SEQ ID No.)10C2G3 1 DP (27-28) (SEQ ID No. 4) 12F3F2 1 DPL (27-29) (SEQ ID No. 5)12F8E4 1 DPL (27-29) (SEQ ID No. 5) 12H4E1 1 EDDPLQ (25-30) (SEQ ID No.6) 6E1B4 1 EDDPLQ (25-30) (SEQ ID No. 6) ^(a)Amino acid sequence of theregion for binding of Prodefensin-A6 to the antibody tested. The numbersbetween parentheses correspond to the position of the epitope on theamino acid sequence of Prodefensin-A6, the numbering beginning at theinitial methionine.

The epitopes recognized by the 1H8C9, 11B2C2, 13E7F3 and 11E8C9antibodies could not be determined by the Spotscan technique, whichindicates that they are not linear. The screening of the phage displaypeptide libraries made it possible to select 5, 3, 7 and 4 mimotopes(linear sequence mimicking an epitope) which react, respectively, withthe 1H8C9, 11B2C2, 11E8C9 and 13E7F3 antibodies. The sequences of thesemimotopes are given in FIG. 4. For each antibody, the sequences of therecognized or immunoreactive mimotopes were aligned in order todetermine a concensus sequence, which is indicated in bold in FIG. 4,which represents the minimum sequence recognized by the antibody. It wasnot possible to find even one of these consensus sequences in theprimary structure (or peptide sequence) of Prodefensin-A6. Thus, theseconsensus sequences correspond to residues dispersed over the primarystructure of the protein but which share a proximity in itsthree-dimensional structure in order to form a conformational epitope.

9. Detection of the Prodefensin-A6 by Sandwich ELISA

9.1. Methodology

The Prodefensin-A6 was detected by sandwich immunoassay using, forexample, the Vidas® ELISA automated system (bioMérieux). This type oftest can also be carried out in a microplate, in an automated or manualmanner. To do this, the ELISA assay was constructed using the reagentsof the Vidas® HBs Ag Ultra kit (bioMérieux, Cat. No. 30315). Thereagents were used as described in the corresponding information sheet(ref. 11728 D-FR-2005/05), with the following modifications:

1. Cones were sensitized with the nine capture antibodies to be tested,at a concentration of 10 μg/ml.

2. The content of the second well of the HBs Ag Ultra cartridge wasreplaced with 300 μl of revealing antibody to be tested (1H8C9 and11E8C9) coupled to biotin, diluted to 1 μg/ml in the buffer (withoutgoat serum) of the second well of the Vidas® HBs Ag Ultra kit.

3. The culture supernatant of 293 Ts transfected with thepCMX-XL5-Prodefensin-A6 plasmid, containing secreted Prodefensin-A6, isadded pure or diluted in PBS directly to the second well of the HBs AgUltra cartridge (50 μl).

4. The ELISA reaction was carried out using the Vidas® automated systemand the HBs Ag Ultra protocol, of which the step of incubating thesample with the capture and revealing antibodies had been brought to 100cycles.

5. The results were obtained in the form of crude values. The signal isin RFV, relative fluorescence value.

9.2. Results

Table 3 reproduces the reactivity of the various combinations ofantibodies on a dilution to 1/2 of the culture supernatant of 293 Tstransfected with the pCMX-XL5-Prodefensin-A6 plasmid and containing thenative Prodefensin-A6 protein.

TABLE 3^(a) Biotinylated detection antibody Capture antibody 1H8C911E8C9 1H8C9 (epitope 2) — 43 11B2D2 (epitope 3)   955* 11 13E7F3(epitope 5)  1088* 2881 11E8C9 (epitope 4)  4015* — 6E1B4 (epitope 1)11646 3407 10C2G3 (epitope 1) 11583 7420 12F3F2 (epitope 1) 11481 644012F8E4 (epitope 1) 11436 3064 12H4E1 (epitope 1) 11868 11037 ^(a)Signalin VIDAS sandwich immunoassay, in RFV (relative fluorescence unit).*signal for supernatant assayed pure. The other supernatants werediluted to ½.

The results presented in Table 3 show that it is possible to detectProdefensin-A6 using sandwich ELISAs based on various combinations ofepitopes: for example, epitopes No. 5 and 4, epitopes No. 1 and 4, orelse preferentially a combination of epitope 2 with any of the otherepitopes identified (1, 3, 4, 5). Even more preferentially, epitope 2 isused for detection. On the other hand, the combination of epitopes No. 2and 4 or else No, 3 and 4 do not make it possible to detectProdefensin-A6.

A finer analysis of the reactivity of the antibodies directed againstepitope 1, used for capture, and combined with the 1H8C9 clone, ispresented in FIG. 5. The 12H4E1 clone is the best capture antibody, andin second position are the 10C2G3 and 12F3F2 clones. The lowest signalsare obtained with the 6E1B4 and 12F8E4 antibodies. This result is verysurprising since the 6E1B4 and 12H4E1 clones recognize the same minimumsequence.

EXAMPLE 3 Serum Assays for the Tumour Markers

1. Patients and Specimens

Blood samples are collected from a network of eight clinical centresdistributed throughout France, in the context of two Huriet-lawprotocols.

In order to obtain serum, the blood sample is taken on a dry tube. Inorder to obtain plasma, the blood sample is taken on an EDTA tube. Aftercoagulation, the tube is centrifuged for 10 min at 1000 g, and the serumis removed, aliquoted and stored at −80° C. The tube of plasma isdirectly centrifuged for 10 min at 1000 g, and the plasma is removed,aliquoted and stored at −80° C. The samples are completely documentedfor the clinical history of the patients.

2. Serum Assay for the Prodefensin-A6 Tumour Marker

The Prodefensin-A6 precursor protein was assayed using the antibodiesdescribed in detail in example 2 and an ELISA assay using the Vidas®automated system (bioMérieux). To do this, the ELISA assay wasconstructed using the reagents of the Vidas® HBs Ag Ultra kit(bioMérieux, Cat. No. 30315). The reagents were used as described in thecorresponding information sheet (ref. 11728 D-FR-2005/05), with thefollowing modifications:

1. The cones were sensitized with the capture antibody 12H4E1 at aconcentration of 15 μg/ml.

2. The content of the second well of the HBs Ag Ultra cartridge wasreplaced with 300 μl of the revealing antibody 1H8C9, coupled to biotin,diluted to 1 μg/ml in the buffer (without goat serum) of the second wellof the Vidas® HBs Ag Ultra kit.

3. The serum or plasma samples (50 μL) were diluted directly in thesecond well of the HBs Ag Ultra cartridge.

4. The ELISA reaction was carried out using the Vidas® automated systemand the HBs Ag Ultra protocol, of which the step of incubating thesample with the capture and revealing antibodies had been brought to 100cycles.

5. The results were obtained in the form of crude values aftersubtraction of the background noise (reading of the substrate beforereaction). A standard curve was established by assaying a range ofconcentrations of the synthetic Prodefensin-A6 peptide (SEQ ID No. 1).The standard curve was plotted by reporting the concentration of thetumour marker along the x-axis and the signal read by Vidas® (RFV orRelative Fluorescence Value) along the y-axis. The concentration oftumour marker present in the body fluid to be assayed (blood, serum,plasma) was calculated by reporting the concentration corresponding tothe RFV signal read by Vidas®.

The results of the serum Prodefensin-A6 assay in the patients by ELISAon a Vidas automated system are given in Table 4. The serumconcentrations of Prodefensin-A6 are between 0 and 10 pg/ml in thepatients having a colorectal adenocarcinoma (CRC+) and between 0 and 2pg/ml in normal individuals (CRC−), which requires an extremelysensitive ELISA assay in order to be able to implement the invention ina biological fluid or remote sample. The only Prodefensin-A6 ELISAassays which make it possible to achieve such a low limit of detectionare based on the combination of antibodies which recognize epitopes 1and 4. It is preferable to capture the Prodefensin-A6 via epitope 1. Thecombination which gives the most sensitive assay is that which was usedhere with the 12H4E1 antibody for capture and the 1H8C9 antibody fordetection.

TABLE 4 Patho- Pro- logical Patient defensin- condition^(a) identifierStage TNM^(b) Age Sex A6 (pg/mL) IDD CLSP046 43 Male 0.40 IDD CLSP049 30Female 1.11 IDD CLSP051 47 Female 0.61 IDD CLSP056 23 Female 2.16 IDDCLSP065 41 Female 1.60 IDD CLSP084 34 Female 0.10 IDD CLSP134 59 Male0.77 IDD CLSP135 47 Female 0.44 IDD CLSP137 35 Female 0.23 IDD CLSP15155 Male 0.44 IDD CLSP152 36 Male 4.06 CRC+ CBSE011 0 TisN0M0 76 Male1.91 CRC+ CLSP059 0 TisN0M0 72 Male 0.49 CRC+ CLSP104 0 TisN0M0 48 Male0.41 CRC+ CBSE001 I T1N0M0 69 Female 1.44 CRC+ CBSE016 I T1N0M0 74 Male2.19 CRC+ CBSE022 I T1N0M0 78 Male 0.29 CRC+ CBSE025 I T2N0M0 81 Female1.45 CRC+ CLSP047 I T1N0M0 76 Female 0.59 CRC+ CLSP062 I T2N0M0 83 Male0.56 CRC+ CLSP067 I T2N0M0 61 Male 0.45 CRC+ CLSP080 I T2N0M0 74 Female1.29 CRC+ CLSP085 I T2N0M0 74 Female 3.26 CRC+ CLSP086 I T2N0M0 61 Male0.23 CRC+ CLSP093 I T2N0M0 71 Male 0.39 CRC+ CLSP100 I T3N0M0 53 Male0.41 CRC+ CLSP118 I T2N0M0 60 Female 0.76 CRC+ CLSP145 I T1N0M0 71 Male1.38 CRC+ CLSP146 I T2N0M0 55 Female 0.24 CRC+ CLSP150 I T2N0M0 61 Male0.24 CRC+ CBSE004 II T3N0M0 85 Male 0.07 CRC+ CBSE017 II T3N0M0 74 Male1.00 CRC+ CBSE018 II T4N0M0 82 Male 0.26 CRC+ CLSP043 II T3N0M0 75 Male1.25 CRC+ CLSP060 II T4N0M0 84 Male 1.26 CRC+ CLSP069 II T3N0M0 46 Male1.07 CRC+ CLSP075 II T3N0M0 65 Male 1.20 CRC+ CLSP087 II T3N0M0 75 Male1.52 CRC+ CLSP088 II T3N0M0 88 Female 0.72 CRC+ CLSP096 II T3N0M0 79Female 0.40 CRC+ CLSP105 II T3N0M0 73 Male 1.61 CRC+ CLSP107 II T3N0M079 Male 0.38 CRC+ CLSP110 II T3N0M0 67 Female 0.39 CRC+ CLSP113 IIT3N0M0 50 Male 0.69 CRC+ CLSP115 II T3N0M0 65 Female 3.62 CRC+ CLSP117II T3N0M0 78 Female 1.62 CRC+ CLSP119 II T3N0M0 78 Male 0.80 CRC+CLSP122 II T3N0MX 54 Male 0.81 CRC+ CLSP133 II T3N0M0 78 Female 0.83CRC+ CLSP136 II T3N0M0 67 Female 0.22 CRC+ CLSP143 II T3N0M0 76 Female1.25 CRC+ CLSP147 II T3N0M0 83 Male 0.77 CRC+ CLSP154 II T3N0M0 76Female 0.86 CRC+ CLSP157 II T3N0 45 Female 0.42 CRC+ GHBD020 II T3N0M075 Male 1.01 CRC+ GHBD023 II T3N0M0 57 Male 0.23 CRC+ GHBD025 II T3N0M073 Male 0.73 CRC+ GHBD029 II T3N0M0 75 Female 2.22 CRC+ CBSE005 IIIT3N1M0 73 Male 4.27 CRC+ CBSE006 III T3N1M0 84 Male 9.24 CRC+ CBSE007III T3N1M0 77 Female 2.02 CRC+ CBSE010 III T4N2M0 67 Male 9.17 CRC+CBSE013 III T3N1M0 82 Male 4.50 CRC+ CBSE023 III T4N2M0 76 Male 2.04CRC+ CLSP044 III T3N1M0 80 Male 0.47 CRC+ CLSP050 III T2N1M0 88 Female2.18 CRC+ CLSP072 III T3N1M0 79 Male 2.17 CRC+ CLSP073 III T2N1M0 77Male 1.59 CRC+ CLSP074 III T3N2M0 79 Female 0.64 CRC+ CLSP089 III T4N2M084 Female 0.81 CRC+ CLSP090 III T1N1M0 65 Female 1.47 CRC+ CLSP091 IIIT3N1M0 55 Male 0.29 CRC+ CLSP094 III T3N1M0 72 Male 0.47 CRC+ CLSP097III T3N1M0 71 Female 0.74 CRC+ CLSP098 III T3N2M0 61 Male 0.69 CRC+CLSP103 III T2N1MX 60 Male 0.40 CRC+ CLSP106 III T4N2M0 85 Female 1.90CRC+ CLSP121 III T3N2M0 76 Male 0.49 CRC+ CLSP123 III T4N1MX 68 Male1.81 CRC+ CLSP138 III T3N1MX 78 Male 1.03 CRC+ CLSP141 III T3N1M0 70Male 1.21 CRC+ CLSP144 III T3N1M0 52 Female 0.93 CRC+ CLSP153 III T3N2MX85 Male 5.09 CRC+ GHBD019 III T3N1M0 74 Male 0.92 CRC+ CBSE012 IV T4N3M137 Female 0.50 CRC+ CBSE019 IV T3N2M1 58 Male 1.52 CRC+ CBSE026 IVT4N1M1 72 Male 0.61 CRC+ CBSE027 IV T4N2M1 78 Female 1.04 CRC+ CLSP042IV T3N2M1 56 Male 1.39 CRC+ CLSP057 IV T3N2M1 61 Female 0.27 CRC+CLSP068 IV TXNXM1 60 Female 1.29 CRC+ CLSP079 IV T3N0M1 81 Female 0.81CRC+ CLSP083 IV T3N1M1 67 Female 0.79 CRC+ CLSP095 IV T3N1M1 64 Male0.65 CRC+ CLSP109 IV T3N1M1 60 Male 0.88 CRC+ CLSP132 IV T3N1M1 62 Male2.80 CRC+ CLSP156 IV T4N0M1 59 Male 1.25 CRC+ CLSP159 IV T3N0M1 68Female 0.89 CRC+ CLSP160 IV TXNXM1 70 Male 2.77 CRC+ CLSP161 IV T3N2M178 Female 0.64 Adenoma CLSP055 73 Male 0.55 Adenoma CLSP058 55 Female0.58 Adenoma CLSP061 61 Female 0.62 Adenoma CLSP099 T0N0M0 62 Male 0.13Adenoma CLSP116 63 Male 0.66 Adenoma CLSP120 56 Female 0.30 AdenomaCLSP142 T0 21 Male 1.77 Adenoma CLSP148 TisN0M0 50 Female 1.07 AdenomaCLSP149 T1N0M0 50 Female 1.69 CRC− N00656 47 Female 0.22 CRC− N006615 43Female 0.45 CRC− N00664- 44 Male 0.13 CRC− N006658 48 Male 0.46 CRC−N009901 52 Male 0.65 CRC− N011147 50 Male 0.87 CRC− N011155 51 Male 0.61CRC− N011243 52 Male 0.33 CRC− N017218 44 Female 0.49 CRC− N017234 37Male 0.47 CRC− N017250 48 Female 0.48 CRC− N017269 40 Male 0.82 CRC−N017365 44 Female 0.54 CRC− N017402 25 Male 0.65 CRC− N017410 37 Male0.53 CRC− N018552 42 Male 0.33 CRC− N041082 58 Male 1.16 CRC− N041138 58Male 0.97 CRC− N044703 54 Male 0.37 CRC− N045730 50 Male 0.57 CRC−N14397- 58 Male 0.71 CRC− N143988 62 Male 0.49 CRC− N144358 61 Female0.67 CRC− N146601 57 Male 0.81 CRC− N14661- 61 Female 1.14 CRC− N14669552 Male 0.91 CRC− N14813- 57 Male 0.72 CRC− N148279 55 Male 1.04 CRC−N148340 51 Male 1.05 CRC− N314164 48 Male 1.00 CRC− N318050 56 Male 0.64CRC− N318077 56 Male 1.02 CRC− N318368 60 Male 0.50 CRC− N318384 58Female 0.69 CRC− N318421 60 Female 0.64 CRC− N325015 42 Male 0.44 CRC−N329630 59 Female 0.39 CRC− N370529 57 Male 0.40 CRC− N376461 58 Male0.26 CRC− N376488 63 Female 0.22 CRC− N37663- 62 Male 0.40 CRC− N37676058 Female 0.57 CRC− N376912 64 Female 0.57 CRC− N418599 56 Female 0.23CRC− N418687 28 Female 0.19 CRC− N418716 53 Female 0.65 CRC− N418740 54Male 0.75 CRC− N418759 49 Female 0.84 CRC− N418804 54 Female 0.24 CRC−N440216 60 Female 0.40 CRC− N440478 60 Female 0.49 CRC− N440507 64 Male1.02 CRC− N469775 36 Male 0.47 CRC− N491028 50 Male 0.59 CRC− N491191 52Female 1.16 CRC− N491247 58 Male 0.35 CRC− N491386 58 Male 0.71 CRC−N491685 56 Male 0.52 CRC− N511463 52 Female 0.46 CRC− N511471 59 Female0.68 CRC− N511498 55 Female 0.58 CRC− N518059 0 Male 0.69 CRC− N51851858 Female 0.78 CRC− N518542 60 Male 0.81 CRC− N519086 59 Male 0.80 CRC−N527135 56 Female 0.39 CRC− N527450 56 Female 0.38 CRC− N557699 55 Male1.40 CRC− N557701 57 Male 1.66 CRC− N557736 56 Male 0.41 CRC− N557760 60Male 1.68 CRC− N593116 52 Female 0.29 CRC− N593167 53 Male 1.12 CRC−N593183 52 Female 0.25 CRC− N593255 51 Male 1.88 CRC− N593351 57 Male1.34 CRC− N744056 51 Male 0.61 CRC− N748022 50 Female 0.33 CRC− N83596645 Male 0.62 CRC− N836299 52 Female 1.13 CRC− N857704 52 Female 0.29CRC− N858037 63 Female 1.15 CRC− N858248 62 Male 1.72 CRC− N862239 53Male 0.24 CRC− N862298 63 Male 0.42 CRC− N862300 51 Male 1.81 ^(a)IDD =Inflammatory bowel diseases (Crohn's disease and Ulcerative colitis)CRC+ = patients having colorectal cancer (adenocarcinoma), CCR− =healthy individuals. ^(b)TNM: stage of tissue invasion (T), lymph nodeinvasion (N) and remote invasion (metastases, M)

The doses obtained for the patients analysed are reported in FIG. 6. Itmay be noted, in this figure and in Table 4, that 2, 2, 10 and 2patients having respectively stage I, II, III or IV colorectal cancershow a clear increase in their amount of serum Prodefensin-A6, strictlyabove the highest value observed in the group of healthy individuals(1.88 pg/ml). In the adenoma group, no amount observed exceeds thisvalue, while in the IDD group, there are 2.

EXAMPLE 4 Use of the Serum Assays for the Tumour Markers in Combination

The applicant showed in example 3 that abnormally elevated amounts ofProdefensin-A6 precursor protein could be observed in the bloodstream ofcertain patients having colorectal cancer. Furthermore, the applicantshowed in patent applications WO2009/024691, WO2009019365, WO2009019368,WO2009019369, WO2009019366, WO2009019370 and WO2009019367 thatabnormally elevated or abnormally reduced amounts of other tumourmarkers, such as LEI, ezrin, aminoacylase-1, L-FABP, Apo A1, Apo A2,I-plastin, beta2-microglobulin, CEA, CA19-9, testosterone, galectin-3,LDH-B, proteasome 20S, E-cadherin or regenerating islet-derived protein3 alpha, otherwise known as pancreatitis associated protein (PAP1),could also be observed in the bloodstream of certain patients havingcolorectal cancer. The methods for assaying these tumour markers weredescribed in the abovementioned patent applications. The method forassaying MIF was carried out with the human MIF Quantikine ELISA kitfrom R&D Systems (Cat No. DMF00) according to the producer'sinstructions.

Surprisingly, the increase or the decrease in the amount, in the blood,of two given markers is not systematically observed in the samepatients. As a result, the combination of several tumour markers makesit possible to increase the number of patients identified as havingcolorectal cancer. Thus, a patient A may present an increase or adecrease in one or more tumour markers (group X), it being possible forsaid markers of group X to be normal in a patient B; in this samepatient B, one or more other tumour markers (group Y) may be elevated orreduced, it being possible for said markers of group Y to be normal inpatient A.

The various tumour markers assayed by the applicant may thus be combinedby means of various mathematical algorithms well known to those skilledin the art. By way of illustration, and without this example beingexhaustive in nature, the following method was carried out:

-   -   1. A threshold value was set for each tumour marker.    -   2. When the amount of the tumour marker in the blood was        increased in the case of colorectal cancer, the amount in the        blood, obtained for a given patient, was divided by its        threshold value. When the amount of the tumour marker in the        blood was decreased in the case of colorectal cancer, the amount        in the blood, obtained for a given patient, was inverted and        then multiplied by its threshold value.    -   3. When the “amount in the blood divided by threshold value”        ratio was greater than 1, the ratio was multiplied by a        coefficient, for example 10. The value thus obtained was named        the “score”, for the patient studied, for the tumour marker        under consideration.    -   4. The scores obtained for various tumour markers were added,        with them being weighted by a factor specific to each marker. In        the case of the example below, all the weighting factors were        set at 1.    -   5. The sum of the scores was divided by the total number of        scores added and the value thus obtained was named the “total        score”.    -   6. The patient is diagnosed as having colorectal cancer when the        total score of said patient is increased relative to a threshold        score.

The total scores for a selection of 2, 3, 4, 5, 7 and 8 markerscomprising Prodefensin-A6 are given in Table 5.

The combination of the Prodefensin-A6 and CEA tumour markers thus makesit possible to obtain, for the same group of 78 patients, increasedtotal scores “2^(a)” in 41 patients having a colorectal adenocarcinoma,whereas assaying Prodefensin-A6 and CEA alone showed an increase,respectively, in 17 and 28 patients only.

The combination of the Prodefensin-A6 and CA19-9 tumour markers thusmakes it possible to obtain, for the same group of 78 patients,increased total scores “2^(b)” in 28 patients having a colorectaladenocarcinoma, whereas assaying Prodefensin-A6 and CEA alone showed anincrease, respectively, in 17 and 15 patients only.

The combination of the Prodefensin-A6 and beta2-microglobulin tumourmarkers thus makes it possible to obtain, for the same group of 78patients, increased total scores “2” in 49 patients having a colorectaladenocarcinoma, whereas assaying Prodefensin-A6 and beta2-microglobulinalone showed an increase, respectively, in 17 and 43 patients only.

The combination of the Prodefensin-A6 and L-FABP tumour markers thusmakes it possible to obtain, for the same group of 78 patients,increased total scores “2” in 41 patients having a colorectaladenocarcinoma, whereas assaying Prodefensin-A6 and L-FABP alone showedan increase, respectively, in 17 and 35 patients only.

The combination of the Prodefensin-A6, CA19-9 and CEA tumour markersthus makes it possible to obtain, for the same group of 78 patients,increased total scores “3” in 46 patients having a colorectaladenocarcinoma, whereas assaying Prodefensin-A6, CA19-9 and CEA aloneshowed an increase, respectively, in 17, 15 and 28 patients only.

The combination of the Prodefensin-A6, beta2-microglobulin and CEAtumour markers thus makes it possible to obtain, for the same group of78 patients, increased total scores “3^(f)” in 60 patients having acolorectal adenocarcinoma, whereas assaying Prodefensin-A6,beta2-microglobulin and CEA alone showed an increase, respectively, in17, 43 and 28 patients only.

The combination of the Prodefensin-A6, beta2-microglobulin, CA19-9 andCEA tumour markers thus makes it possible to obtain, for the same groupof 78 patients, increased total scores “4^(g)” in 63 patients having acolorectal adenocarcinoma, whereas assaying Prodefensin-A6,beta2-microglobulin, CA19-9 and CEA alone showed an increase,respectively, in 17, 43, 15 and 28 patients only.

The combination of the Prodefensin-A6, beta2-microglobulin, L-FABP andCEA tumour markers thus makes it possible to obtain, for the same groupof 78 patients, increased total scores “4^(h)” in 69 patients having acolorectal adenocarcinoma, whereas asssaying Prodefensin-A6,beta2-microglobulin, L-FABP and CEA alone showed an increase,respectively, in 17, 43, 35 and 28 patients only.

The combination of the Prodefensin-A6, CA19-9, L-FABP and CEA tumourmarkers thus makes it possible to obtain, for the same group of 78patients, increased total scores “4^(i)” in 59 patients having acolorectal adenocarcinoma, whereas asssaying Prodefensin-A6, CA19-9,L-FABP and CEA alone showed an increase, respectively, in 17, 15, 35 and28 patients only.

The combination of the Prodefensin-A6, beta2-microglobulin, CA19-9,L-FABP and CEA tumour markers thus makes it possible to obtain, for thesame group of 78 patients, increased total scores “5^(j)” in 71 patientshaving a colorectal adenocarcinoma, whereas assaying Prodefensin-A6,beta2-microglobulin, CA19-9, L-FABP and CEA alone showed an increased,respectively, in 17, 43, 15, 35 and 28 patients only.

The combination of the Prodefensin-A6, beta2-microglobulin, CA19-9,galectin-3, L-FABP, MIF and CEA tumour markers thus makes it possible toobtain, for the same group of 78 patients, increased total scores“7^(k)” in 74 patients having a colorectal adenocarcinoma, whereasassaying Prodefensin-A6, beta2-microglobulin, CA19-9, galectin-3,L-FABP, MIF and CEA alone showed an increase, respectively, in 17, 43,15, 13, 35, 23 and 28 patients only.

The combination of the Prodefensin-A6, beta2-microglobulin, CA19-9,galectin-3, L-FABP, MIF, I-plastin and CEA tumour markers thus makes itpossible to obtain, for the same group of 78 patients, increased totalscores “8^(l)” in 75 patients having a colorectal adenocarcinoma,whereas assaying Prodefensin-A6, beta2-microglobulin, CA19-9,galectin-3, L-FABP, MIF, I-plastin and CEA alone showed an increase,respectively, in 17, 43, 15, 13, 35, 23, 3 and 28 patients only.

TABLE 5 Pathological Patient Score Score Score Score Score Score ScoreScore Score Score Score Score condition identifier 2^(a) 2^(b) 2^(c)2^(d) 3^(e) 3^(f) 4^(g) 4^(h) 4^(i) 5^(j) 7^(k) 8^(l) CRC− N011155 0.320.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 CRC− N011243 0.180.18 0.18 0.14 0.18 0.18 0.18 0.14 0.14 0.14 0.14 0.14 CRC− N14661- 0.610.61 0.61 0.39 0.61 0.61 0.61 0.39 0.39 0.39 0.39 0.39 CRC− N044703 0.200.20 0.20 0.21 0.20 0.20 0.20 0.21 0.21 0.21 0.21 0.21 CRC− N318050 0.340.34 0.34 0.31 0.34 0.34 0.34 0.31 0.31 0.31 0.31 0.31 CRC− N418599 0.120.12 0.12 0.23 0.12 0.12 0.12 0.23 0.23 0.23 0.23 0.23 CRC− N017410 0.280.28 0.28 0.27 0.28 0.28 0.28 0.27 0.27 0.27 0.27 0.27 CRC− N329630 0.210.21 0.21 0.33 0.21 0.21 0.21 0.33 0.33 0.33 0.33 0.33 CRC− N045730 0.300.30 0.30 0.32 0.30 0.30 0.30 0.32 0.32 0.32 0.32 0.32 CRC− N593116 0.150.15 0.15 0.27 0.15 0.15 0.15 0.27 0.27 0.27 0.27 0.27 CRC− N018552 0.180.18 0.18 0.27 0.18 0.18 0.18 0.27 0.27 0.27 0.27 0.27 CRC− N017218 0.260.26 0.26 0.38 0.26 0.26 0.26 0.38 0.38 0.38 0.38 0.38 CRC− N011147 0.460.46 0.46 0.43 0.46 0.46 0.46 0.43 0.43 0.43 0.43 0.43 CRC− N017365 0.290.29 0.29 0.33 0.29 0.29 0.29 0.33 0.33 0.33 0.33 0.33 CRC− N440216 0.210.21 0.21 0.33 0.21 0.21 0.21 0.33 0.33 0.33 0.33 0.33 CRC− N376912 0.300.30 0.30 0.38 0.30 0.30 0.30 0.38 0.38 0.38 0.38 0.38 CRC− N527450 0.200.20 0.20 0.34 0.20 0.20 0.20 0.34 0.34 0.34 0.34 0.34 CRC− N748022 0.180.18 0.18 0.26 0.18 0.18 0.18 0.26 0.26 0.26 0.26 0.26 CRC− N593183 0.130.13 0.13 0.32 0.13 0.13 0.13 0.32 0.32 0.32 0.32 0.32 CRC− N009901 0.350.35 0.35 0.46 0.35 0.35 0.35 0.46 0.46 0.46 0.46 0.46 CRC− N017269 0.440.44 0.44 0.49 0.44 0.44 0.44 0.49 0.49 0.49 0.49 0.49 CRC− N376461 0.140.14 0.14 0.37 0.14 0.14 0.14 0.37 0.37 0.37 0.37 0.37 CRC− N862300 0.960.96 0.96 0.77 0.96 0.96 0.96 0.77 0.77 0.77 0.77 0.77 CRC− N376488 0.120.12 0.12 0.42 0.12 0.12 0.12 0.42 0.42 0.42 0.42 0.42 CRC− N017234 0.250.25 0.25 0.47 0.25 0.25 0.25 0.47 0.47 0.47 0.47 0.47 CRC− N527135 0.210.21 0.21 0.44 0.21 0.21 0.21 0.44 0.44 0.44 0.44 0.44 CRC− N440478 0.260.26 0.26 0.44 0.26 0.26 0.26 0.44 0.44 0.44 0.44 0.44 CRC− N017402 0.350.35 0.35 0.56 0.35 0.35 0.35 0.56 0.56 0.56 0.56 0.56 CRC− N491191 0.620.62 0.60 0.62 0.62 0.60 0.60 0.60 0.62 0.60 0.55 0.55 CRC− N835966 0.330.33 0.56 0.25 0.33 0.56 0.56 0.43 0.25 0.43 0.45 0.45 CRC− N017250 0.210.22 0.26 0.29 0.20 0.21 0.20 0.25 0.23 0.23 0.23 0.23 CRC− N144358 0.360.36 0.49 0.28 0.36 0.49 0.49 0.39 0.28 0.39 0.40 0.40 CRC− N143988 0.260.26 0.51 0.33 0.26 0.51 0.51 0.47 0.33 0.47 0.48 0.48 CRC− N511498 0.300.23 0.51 0.35 0.25 0.44 0.37 0.43 0.29 0.37 0.34 0.33 CRC− N318384 0.440.19 0.49 0.31 0.30 0.50 0.38 0.44 0.29 0.35 0.37 0.33 CRC− N148340 0.320.33 0.62 0.54 0.24 0.44 0.35 0.46 0.32 0.39 0.42 0.39 CRC− N491386 0.200.20 0.48 0.44 0.14 0.32 0.25 0.37 0.23 0.30 0.33 0.29 CRC− N518518 0.490.29 0.59 0.48 0.38 0.59 0.48 0.57 0.42 0.49 0.44 0.39 CRC− N518542 0.320.22 0.57 0.22 0.22 0.45 0.34 0.34 0.16 0.27 0.28 0.29 CRC− N858248 0.610.63 0.87 0.72 0.52 0.68 0.60 0.65 0.53 0.59 0.55 0.48 CRC− N491247 0.350.19 0.42 0.35 0.30 0.46 0.39 0.47 0.36 0.42 0.43 0.39 CRC− N148279 0.380.35 0.70 0.46 0.30 0.53 0.44 0.49 0.32 0.42 0.44 0.40 CRC− N418687 0.260.06 0.45 0.23 0.18 0.44 0.33 0.42 0.22 0.34 0.41 0.36 CRC− N857704 0.220.33 0.44 0.22 0.32 0.39 0.42 0.37 0.31 0.39 0.46 0.41 CRC− N593255 0.650.54 0.97 0.72 0.46 0.74 0.58 0.67 0.45 0.55 0.50 0.43 CRC− N862298 0.370.15 0.22 0.22 0.27 0.37 0.27 0.37 0.27 0.27 0.41 0.33 CRC− N318421 0.580.21 0.67 0.47 0.41 0.72 0.56 0.69 0.46 0.57 0.56 0.49 CRC− N511471 0.290.21 0.53 0.47 0.22 0.43 0.33 0.46 0.31 0.38 0.38 0.36 CRC− N325015 0.400.48 0.50 0.33 0.51 0.52 0.58 0.50 0.49 0.55 0.54 0.47 CRC− N146695 0.270.30 0.54 0.46 0.22 0.38 0.32 0.39 0.27 0.34 0.36 0.32 CRC− N318077 0.470.32 0.64 0.59 0.35 0.56 0.44 0.58 0.42 0.48 0.51 0.46 CRC− N858037 0.400.32 0.71 0.50 0.28 0.53 0.41 0.49 0.30 0.40 0.47 0.42 CRC− N511463 0.160.20 0.46 0.29 0.16 0.33 0.29 0.34 0.20 0.30 0.43 0.40 CRC− N593167 0.450.62 0.59 0.47 0.51 0.49 0.53 0.46 0.47 0.50 0.50 0.55 CRC− N557736 0.570.14 0.41 0.23 0.40 0.58 0.45 0.50 0.36 0.41 0.41 0.39 CRC− N314164 0.360.46 0.58 0.56 0.37 0.45 0.43 0.48 0.42 0.46 0.54 0.47 CRC− N862239 0.310.29 0.44 0.24 0.36 0.46 0.46 0.43 0.36 0.44 0.46 0.40 CRC− N491685 0.260.17 0.64 0.42 0.20 0.51 0.40 0.52 0.29 0.43 0.49 0.44 CRC− N518059 0.270.22 0.55 0.58 0.20 0.42 0.34 0.52 0.35 0.43 0.47 0.48 CRC− N519086 0.310.32 0.56 0.52 0.28 0.44 0.38 0.48 0.36 0.43 0.43 0.45 CRC− N37663- 0.330.61 0.55 0.35 0.55 0.52 0.64 0.51 0.54 0.61 0.62 0.55 CRC− N440507 0.430.28 0.76 0.54 0.30 0.61 0.47 0.59 0.36 0.48 0.53 0.46 CRC− N376760 0.650.52 0.53 0.43 0.68 0.69 0.70 0.65 0.65 0.67 0.64 0.59 CRC− N469775 0.240.17 0.50 0.39 0.20 0.41 0.33 0.44 0.28 0.37 0.47 0.41 CRC− N491028 0.260.20 0.54 0.35 0.20 0.43 0.34 0.42 0.25 0.35 0.44 0.39 CRC− N146601 0.510.42 0.63 0.48 0.47 0.61 0.56 0.59 0.49 0.55 0.62 0.54 CRC− N557701 0.550.68 0.83 0.75 0.52 0.63 0.59 0.63 0.55 0.59 0.57 0.50 CRC− N557760 0.560.56 0.80 0.95 0.45 0.60 0.51 0.70 0.58 0.61 0.58 0.56 CRC− N557699 0.600.72 0.82 0.56 0.63 0.70 0.70 0.62 0.57 0.63 0.63 0.61 CRC− N593351 0.760.48 0.63 0.59 0.59 0.68 0.58 0.63 0.56 0.55 0.55 0.61 CRC− N744056 0.300.48 0.47 0.28 0.42 0.41 0.47 0.36 0.37 0.42 0.49 0.42 CRC+ CBSE011 5.135.12 12.17 5.43 3.45 8.15 6.13 6.29 2.76 5.05 6.17 5.30 CRC+ CLSP0590.35 0.18 0.44 5.71 0.27 0.44 0.35 3.12 2.99 2.51 3.45 3.04 CRC+ CLSP1040.27 7.10 0.44 0.48 4.85 0.40 3.80 0.49 3.82 3.19 5.63 4.96 CRC+ CBSE0010.77 0.77 0.77 10.28 0.77 0.77 0.77 10.28 10.28 10.28 10.28 10.28 CRC+CBSE016 19.26 5.83 12.92 6.18 12.85 17.57 13.18 13.35 9.81 10.69 9.027.73 CRC+ CBSE022 0.20 0.08 8.62 0.38 0.14 5.83 4.38 4.53 0.26 3.62 6.915.93 CRC+ CBSE025 0.72 0.50 8.93 0.87 0.55 6.18 4.69 4.87 0.65 3.94 5.584.80 CRC+ CLSP047 0.29 0.17 5.46 0.37 0.20 3.73 2.80 2.90 0.26 2.33 1.841.66 CRC+ CLSP062 0.37 5.28 6.47 0.40 3.67 4.46 5.91 3.47 2.88 4.83 5.234.66 CRC+ CLSP067 0.21 0.14 0.54 7.28 0.16 0.42 0.33 3.90 3.70 3.13 2.402.16 CRC+ CLSP080 0.40 0.46 5.77 0.69 0.35 3.89 2.97 3.89 0.35 2.97 2.191.88 CRC+ CLSP085 8.75 8.75 9.14 9.00 5.89 6.15 4.65 4.78 4.58 3.86 2.952.61 CRC+ CLSP086 168.1 0.24 0.35 0.31 112.2 112.3 84.30 84.34 84.2967.54 48.35 42.34 CRC+ CLSP093 0.24 0.26 0.42 8.93 0.26 0.37 0.36 4.694.61 3.81 2.85 2.52 CRC+ CLSP100 0.44 0.19 6.86 7.48 0.35 4.79 3.63 7.283.95 5.86 4.32 3.79 CRC+ CLSP118 0.21 0.22 0.63 0.44 0.15 0.42 0.33 0.440.23 0.36 0.40 2.32 CRC+ CLSP145 0.51 0.42 7.10 0.78 0.38 4.83 3.65 3.830.49 3.09 2.41 2.11 CRC+ CLSP146 0.17 0.23 0.36 7.47 0.22 0.31 0.32 3.943.87 3.22 3.99 3.50 CRC+ CBSE004 0.37 0.27 7.18 0.18 0.41 5.02 3.89 3.850.39 3.18 9.53 8.19 CRC+ CBSE017 17.51 0.36 7.89 0.74 11.73 16.76 12.6112.80 9.03 10.28 8.72 7.47 CRC+ CBSE018 0.19 0.17 0.42 0.26 0.19 0.360.32 0.36 0.24 0.33 2.80 2.40 CRC+ CLSP043 0.54 0.56 5.38 0.66 0.51 3.722.90 3.72 0.51 2.90 4.01 3.45 CRC+ CLSP060 0.64 0.66 8.88 6.69 0.65 6.134.76 7.77 3.66 6.35 5.39 4.66 CRC+ CLSP087 0.59 0.45 7.11 0.66 0.43 4.873.67 3.78 0.45 3.04 2.32 2.03 CRC+ CLSP088 13.90 7.95 0.67 5.87 14.449.58 11.07 10.03 13.67 11.13 8.01 7.01 CRC+ CLSP096 19.94 0.29 8.65 0.4413.42 18.99 14.34 14.41 10.23 11.60 8.42 7.37 CRC+ CLSP105 23.90 0.887.46 0.73 16.23 20.62 15.69 15.61 12.32 12.67 10.82 9.47 CRC+ CLSP1070.44 0.47 0.36 5.91 0.54 0.47 0.54 3.26 3.31 2.75 2.07 1.83 CRC+ CLSP1130.31 0.29 0.49 8.43 0.28 0.41 0.36 4.43 4.33 3.59 2.66 2.33 CRC+ CLSP1159.90 9.72 18.17 26.81 6.66 12.29 9.27 17.81 13.59 14.29 11.93 10.44 CRC+CLSP117 0.75 0.57 8.98 7.36 0.60 6.20 4.72 8.12 3.92 6.55 6.25 5.48 CRC+CLSP119 0.22 0.26 7.33 5.92 0.18 4.89 3.69 6.52 2.99 5.24 3.94 3.47 CRC+CLSP122 0.66 0.40 0.68 0.47 0.56 0.75 0.65 0.69 0.55 0.62 3.60 3.15 CRC+CLSP136 0.26 0.25 0.47 0.48 0.31 0.45 0.43 0.55 0.44 0.52 0.62 0.56 CRC+CLSP143 7.99 0.40 0.80 0.82 5.37 5.64 4.26 4.47 4.27 3.60 2.69 2.37 CRC+CLSP147 0.39 0.22 8.75 0.48 0.27 5.96 4.48 4.60 0.34 3.69 4.34 3.81 CRC+CLSP154 9.94 0.43 6.34 0.49 6.76 10.70 8.13 8.16 5.20 6.60 4.93 4.31CRC+ GHBD020 0.32 0.38 5.61 0.76 0.29 3.78 2.89 3.08 0.46 2.51 1.93 1.72CRC+ GHBD025 0.27 0.25 6.97 0.46 0.21 4.69 3.55 3.65 0.29 2.94 2.29 2.00CRC+ GHBD029 5.94 6.09 6.28 6.27 4.08 4.21 3.25 3.34 3.24 2.74 2.09 1.82CRC+ CBSE005 11.39 11.65 17.84 18.52 7.78 11.91 9.07 12.51 9.42 10.1212.87 11.28 CRC+ CBSE006 24.67 24.68 49.15 24.98 16.52 24.67 16.52 16.7112.59 12.59 12.59 10.10 CRC+ CBSE007 5.49 5.45 10.74 25.72 3.71 5.493.71 17.23 12.96 12.96 12.96 10.38 CRC+ CBSE010 30.57 73.65 30.99 32.3553.22 24.78 43.22 22.56 43.90 37.76 34.94 29.99 CRC+ CBSE013 12.31 17.5220.52 18.49 11.91 13.91 13.20 13.69 12.19 13.17 12.77 10.95 CRC+ CBSE0235.78 5.68 5.84 5.83 4.02 4.13 3.22 3.30 3.22 2.74 2.42 2.11 CRC+ CLSP044103.9 0.25 0.53 0.29 69.37 69.55 52.23 52.25 52.11 41.85 30.04 26.32CRC+ CLSP050 5.97 5.86 13.93 11.14 4.03 9.40 7.09 9.73 5.70 7.81 7.776.84 CRC+ CLSP072 11.33 14.86 14.32 11.06 13.61 13.25 14.48 12.58 12.8513.70 9.96 8.73 CRC+ CLSP073 0.67 14.22 7.20 6.00 9.65 4.96 10.62 6.5110.02 10.73 7.76 6.81 CRC+ CLSP074 0.29 0.41 6.82 0.66 0.36 4.63 3.593.71 0.51 3.07 2.31 2.07 CRC+ CLSP089 25.65 0.62 9.83 0.41 17.37 23.5117.84 17.73 13.13 14.35 10.39 9.11 CRC+ CLSP091 5.55 0.27 0.39 6.43 3.833.91 3.03 6.11 6.05 4.96 3.71 3.26 CRC+ CLSP094 6.67 0.36 0.25 0.31 4.616.67 4.61 4.57 3.54 3.54 2.89 2.44 CRC+ CLSP097 0.31 0.30 0.64 0.55 0.270.50 0.42 0.55 0.38 0.48 0.46 0.40 CRC+ CLSP106 5.05 5.13 12.36 11.503.42 8.24 6.22 9.40 5.78 7.55 5.53 4.84 CRC+ CLSP121 0.22 0.23 0.51 0.410.21 0.40 0.35 0.44 0.30 0.39 1.93 1.69 CRC+ CLSP123 0.63 0.62 9.0314.97 0.51 6.12 4.66 11.83 7.63 9.52 11.26 9.85 CRC+ CLSP138 9.60 0.308.82 0.56 6.42 12.10 9.09 9.22 4.96 7.39 5.43 4.76 CRC+ CLSP141 0.430.38 0.63 10.19 0.32 0.49 0.40 5.30 5.18 4.27 3.31 2.90 CRC+ CLSP15333.40 23.59 21.96 29.87 28.97 27.88 25.93 29.08 29.89 27.28 19.56 17.12CRC+ GHBD019 7.06 0.24 0.68 0.60 4.71 5.00 3.75 3.93 3.71 3.14 2.34 2.08CRC+ CBSE012 5.81 12.11 0.47 0.30 11.85 4.10 9.06 3.16 8.98 7.32 6.195.30 CRC+ CBSE019 6.40 0.63 0.88 7.05 4.42 4.58 3.55 6.76 6.64 5.50 4.714.04 CRC+ CBSE026 195.7 119.8 8.71 6.43 210.2 136.2 161.9 105.3 160.8132.1 95.87 83.92 CRC+ CBSE027 175.0 0.55 5.36 5.79 175.0 120.0 120.092.78 120.3 92.78 68.71 61.25 CRC+ CLSP042 153.8 0.76 5.43 8.15 102.8105.9 79.62 83.31 80.98 66.81 50.00 43.77 CRC+ CLSP057 15.32 0.19 0.4010.84 10.30 10.44 7.89 13.21 13.11 10.62 12.94 12.80 CRC+ CLSP068 337.0120.0 5.60 7.64 304.4 228.2 231.0 174.8 232.0 187.7 138.3 121.2 CRC+CLSP079 0.43 25.92 0.59 0.60 25.92 0.59 17.53 0.65 17.54 13.34 9.16 7.91CRC+ CLSP083 0.37 0.46 0.46 0.54 0.41 0.42 0.43 0.48 0.47 0.48 0.44 0.39CRC+ CLSP095 150.8 0.59 0.59 5.56 100.8 100.8 75.81 78.29 78.29 62.8046.47 40.69 CRC+ CLSP109 0.47 9.79 0.63 34.00 9.79 0.63 6.79 22.93 29.0421.97 17.37 14.96 CRC+ CLSP132 7.74 7.84 15.99 17.61 5.42 10.86 8.3413.23 9.15 10.74 9.52 8.36 CRC+ CLSP156 45.79 24.26 8.49 0.73 46.4735.97 38.94 27.17 35.05 31.31 28.46 24.92 CRC+ CLSP159 67.36 33.80 6.710.66 67.28 49.22 53.70 37.13 50.67 43.13 32.70 28.61 CRC+ CLSP160 7.867.49 7.73 42.10 5.32 5.48 4.17 21.48 21.36 17.23 12.46 10.90 CRC+CLSP161 18.00 0.52 8.72 0.61 12.23 17.70 13.45 13.49 9.40 10.94 7.996.99 Threshold 0.96 0.96 0.97 0.95 0.96 0.96 0.96 0.77 0.77 0.77 0.770.77 Specificity (%) 100 100 100 100 100 100 100 100 100 100 100 100Sensitivity (%) 52.56 35.90 62.82 89.13 58.97 76.92 80.77 88.46 75.6491.03 94.87 96.15 CRC+ above the 41 28 49 41 46 60 63 69 59 71 74 75threshold Score 2^(a): combination of CEA and Prodefensin-A6 Score2^(b): combination of CA19-9 and Prodefensin-A6 Score 2^(c): combinationof beta2-microglobulin and Prodefensin-A6 Score 2^(d): combination ofL-FABP and Prodefensin-A6 Score 3^(e): combination of CEA, CA19-9 andProdefensin-A6 Score 3^(f): combination of CEA, beta2-microglobulin andProdefensin-A6 Score 4^(g): combination of CEA, beta2-microglobulin,CA19-9 and Prodefensin-A6 Score 4^(h): combination of CEA,beta2-microglobulin, L-FABP and Prodefensin-A6 Score 4^(i): combinationof CEA, CA19-9, L-FABP and Prodefensin-A6 Score 5^(j): combination ofCEA, beta2-microglobulin, CA19-9, L-FABP and Prodefensin-A6 Score 7^(k):combination of CEA, beta2-microglobulin, CA19-9, galectin-3,Prodefensin-A6, L-FABP and MIF Score 8^(l): combination of CEA,beta2-microglobulin, CA19-9, Prodefensin-A6, galectin-3, L-FABP, MIF andI-plastin CRC+ = patients having colorectal cancer (adenocarcinoma),CRC− = healthy individuals.

EXAMPLE 5 Detection of the Tumour Markers by Means of the LC-MRM-MSTechnique

1. Methodology

In order to be able to decrease the detection limit to a few ng/ml, animproved MRM-MS method was used. The successive steps of this methodare: 1) immunodepletion of the abundant proteins, 2) trypsin digestion,3) SPE (solid-phase extraction) fractionation of the peptides, 4) liquidchromatography (LC) coupled to MRM-MS.

The setting up was carried out on spike samples by adding theProdefensin-A6 synthetic peptide (SEQ ID No. 1).

Immunodepletion.

The depletion of the abundant proteins in the serum was carried outusing the commercial Vivapure anti-HSA kit from Vivascience.Alternatively, the Proteoextract Albumin/IgG kit from Calbiochem and theAurum™ serum Protein Minikit from Bio-Rad were also used. It is alsopossible to produce the specific resins in the laboratory, by coupling amonoclonal antibody directed against the protein to be depleted, to aCNBr-activated Sepharose 4B resin (Amersham Bioscience), according tothe producer's instructions.

Enzymatic Digestion.

The depleted serum samples are denatured in a 6M urea solution bufferedwith 10 mM of Tris, pH 8, and containing 30 mM of dithiothreitol, for 40minutes at 40° C., and then alkylated with 50 mM iodoacetamide, atambient temperature for 40 minutes, in the dark. They are dilutedsix-fold in water, and the trypsin digestion is then carried out at 37°C. overnight, using an enzyme/substrate ratio of 1/30 (Promega). Thedigestion is stopped by adding formic acid at a final concentration of0.5%. The digested samples are desalified by solid-phase extraction(SPE) using the Oasis HLB 3 cc reverse-phase cartridges (60 mg)(Waters). After application of the sample, the cartridges are washedwith 1 ml of formic acid at 0.1%, and then the elution is carried outwith a methanol/water mixture (80/20 v/v) containing 0.1% of formicacid. The eluates are dried under vacuum.

SPE Fractionation.

The dry samples are taken up in 1 ml of acetate buffer and loaded ontoOasis MCX (mixed cation exchange) 60 mg mixed cartridges (hydrophobicand cation exchange) (Waters) pre-equilibrated in acetate buffer andmethanol. The cartridges are washed with 1 ml of acetate buffer and 1 mlof methanol. The peptides of interest (Table 6) are eluted with 1 ml ofa methanol/acetate buffer mixture (50/50 v/v). The pH of the acetatebuffer is chosen according to the isoelectric point of the peptide ofinterest. The eluates are dried under vacuum, and dissolved in 200 μl ofa solution of acetonitrile/water (3/97 v/v) containing 0.1% of formicacid. A 50 μl aliquot was injected into the LC coupled to an MS-MSsystem.

Liquid Chromatography and Mass Spectrometry.

The LC-MS analysis was carried out on an HP 1100 series high pressurechromatographic system (HPLC) with a binary pump and injector (AgilentTechnologies), coupled to a mass spectrometer, either a Sciex API 2000triple quadripole or a Sciex API 4000 Qtrap (hybrid triplequadripole-ion trap MS) (MDS Sciex) for better sensitivity. The LCseparation was carried out on a C₁₈ Symmetry column (Waters), at anelution flow rate of 300 μl/min (eluent A=0.1% formic acid in water,eluent B=0.1% formic acid in acetonitrile, linear gradient of 5% B to50% B in 25 min, then of 50% B to 100% B in 3 min). The MS analysis iscarried out in the positive ionization mode at a voltage of 5500 V,applied as a needle voltage, enabling ionization in the source. Theinstrument verification and the data acquisition are carried out withthe Analyst 1.4.1 software. The nebulizing gas (air) and curtain gas(nitrogen) flows are 30 and 20 psi, respectively. The Turbo V™ ionsource is adjusted to 400° C., the auxiliary nitrogen flow to 40 psi.The MRM transitions recorded for each peptide are reproduced in Table 6.The collision energy (CE), the declustering potential (DP) and thecollision cell exit potential (CXP) are optimized for each of the MRMtransitions selected.

2. Results

The list of theoretical MRM transitions of the sequence SEQ ID No. 1 wasgenerated using the MIDAS (MRM-initiated Detection and Sequencing)software. This list comprises all the double-charged or triple-chargedparent ions of the theoretical tryptic peptides in a mass range of from800 to 3000 Da and all the possible ion fragments of y or b type. Foreach protein, each possible transition was tested in order to determinethe most sensitive and most specific transitions. The result of thisselection is reproduced in Table 6. An example of optimization of theSPE step for transition 727/556 of the peptide EPLQAEDDPLQAK (SEQ ID No.30) is given in FIG. 7. The MCX chromatographic separation was carriedout at various pHs, the pH selected for the rest of the experimentsbeing the pH which makes it possible to obtain the highest area of thepeak.

Furthermore, using a heavy peptide of AQUA type (Sigma) or alternativelya heavy recombinant protein that will serve as an assay standard, it ispossible to quantify, in an absolute manner, the tumour marker ofinterest in a complex biological medium.

TABLE 6 Prodefensin-A6 Sequence (SEQ ID No.) pI Q1 Q3 DP CE CXPEPLQAEDDPLQAK 3.57 727.4 556.4  50 40 28 (SEQ ID No. 30) 218.2  50 35  8915.4  50 30 25 986.4  50 35 27 AYEADAQEQR 3.93 590.8 746.4 100 30 11(SEQ ID No. 31) 817.4 100 30 18 631.3 100 30 11 560.3 100 30 11 946.4100 35 20

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The invention claimed is:
 1. A monoclonal anti-Prodefensin-A6 antibodythat (i) is specific for the propeptide of the Prodefensin-A6 protein,or (ii) recognizes an epitope including an amino acid sequence selectedfrom the group consisting of the amino acid sequences of: (a)X₁X₂X₃X₄X₅X₆X₇X₈R (SEQ ID NO: 7), in which X₁ is V or L, X₂ is T or L,X₃ is P, S or C, X₄ is P or S, X₅ is W or T, X₆ is A, Q, M, C or E, X₇is I, E or D and X₈ is F, Y, S or L; (b) X₁X₂X₃X₄X₅X₆HX₇ (SEQ ID NO:13), in which X₁ is S or T, X₂ is C or absent, X₃ is T, L or E, X₄ is Hor R, X₅ is I, F or E, X₆ is G or V and X₇ is C or N; (c)X₁HPX₂X₃X₄X₅X₆X₇ (SEQ ID NO: 17), in which X₁ is P or W, X₂ is W or E,X₃ is S, A, Q or W, X₄ is M, L, R or P, X₅ is H, F, W or G, X₆ is V or Aand X₇ is I or V; and (d) X₁HX₂X₃X₄X₅ (SEQ ID NO: 22), in which X₁ is Yor N, X₂ is E, D or Q, X₃ is T, N, R, M or K, X₄ is W, H or F and X₅ isP or G.
 2. The monoclonal antibody of claim 1 that is specific for thepropeptide of the Prodefensin-A6 protein.
 3. The monoclonal antibody ofclaim 1 that recognizes an epitope including an amino acid sequenceselected from the group consisting of the amino acid sequences of SEQ IDNOS: 4-6.
 4. The monoclonal antibody of claim 1 that recognizes theepitope including the amino acid sequence of X₁X₂X₃X₄X₅X₆X₇X₈R (SEQ IDNO: 7), in which X₁ is V or L, X₂ is T or L, X₃ is P, S or C, X₄ is P orS, X₅ is W or T, X₆ is A, Q, M, C or E, X₇ is I, E or D and X₈ is F, Y,S or L.
 5. The monoclonal antibody of claim 1 that recognizes theepitope including the amino acid sequence of X₁X₂X₃X₄X₅X₆HX₇ (SEQ ID NO:13), in which X₁ is S or T, X₂ is C or absent, X₃ is T, L or E, X₄ is Hor R, X₅ is I, F or E, X₆ is G or V and X₇ is C or N.
 6. The monoclonalantibody of claim 1 that recognizes the epitope including the amino acidsequence of X₁HPX₂X₃X₄X₅X₆X₇ (SEQ ID NO: 17), in which X₁ is P or W, X₂is W or E, X₃ is S, A, Q or W, X₄ is M, L, R or P, X₅ is H, F, W or G,X₆ is V or A and X₇ is I or V.
 7. The monoclonal antibody of claim 1that recognizes the epitope including the amino acid sequence ofX₁HX₂X₃X₄X₅ (SEQ ID NO: 22), in which X₁ is Y or N, X₂ is E, D or Q, X₃is T, N, R, M or K, X₄ is W, H or F and X₅ is P or G.
 8. The monoclonalantibody of claim 1 that recognizes an epitope including an amino acidsequence selected from the group consisting of the amino acid sequencesof SEQ ID NOS: 8-12.
 9. The monoclonal antibody of claim 1 thatrecognizes an epitope including an amino acid sequence selected from thegroup consisting of the amino acid sequences of SEQ ID NOS: 14-16. 10.The monoclonal antibody of claim 1 that recognizes an epitope includingan amino acid sequence selected from the group consisting of the aminoacid sequences of SEQ ID NOS: 18-21.
 11. The monoclonal antibody ofclaim 1 that recognizes an epitope including an amino acid sequenceselected from the group consisting of the amino acid sequences of SEQ IDNOS: 23-29.
 12. The monoclonal antibody of claim 1 attached to a labelcapable of producing a detectable signal.
 13. The monoclonal antibody ofclaim 1 attached to a solid support.
 14. The monoclonal antibody ofclaim 1 attached to an imaging tracer capable of producing a detectablesignal.
 15. An assay system comprising: the monoclonal antibody of claim3 as a first monoclonal antibody; and a second monoclonal antibody thatrecognizes an epitope including an amino acid sequence selected from thegroup consisting of the amino acid sequences of: (a) X₁X₂X₃X₄X₅X₆X₇X₈R(SEQ ID NO: 7), in which X₁ is V or L, X₂ is T or L, X₃ is P, S or C, X₄is P or S, X₅ is W or T, X₆ is A, Q, M, C or E, X₇ is I, E or D and X₈is F, Y, S or L; (b) X₁X₂X₃X₄X₅X₆HX₇ (SEQ ID NO: 13), in which X₁ is Sor T, X₂ is C or absent, X₃ is T, L or E, X₄ is H or R, X₅ is I, F or E,X₆ is G or V and X₇ is C or N; (c) X₁HPX₂X₃X₄X₅X₆X₇ (SEQ ID NO: 17), inwhich X₁ is P or W, X₂ is W or E, X₃ is S, A, Q or W, X₄ is M, L, R orP, X₅ is H, F, W or G, X₆ is V or A and X₇ is I or V; and (d)X₁HX₂X₃X₄X₅ (SEQ ID NO: 22), in which X₁ is Y or N, X₂ is E, D or Q, X₃is T, N, R, M or K, X₄ is W, H or F and X₅ is P or G.
 16. A kitcomprising: the monoclonal antibody of claim 3 as a first monoclonalantibody; and a second monoclonal antibody that recognizes an epitopeincluding an amino acid sequence selected from the group consisting ofthe amino acid sequences of: (a) X₁X₂X₃X₄X₅X₆X₇X₈R (SEQ ID NO: 7), inwhich X₁ is V or L, X₂ is T or L, X₃ is P, S or C, X₄ is P or S, X₅ is Wor T, X₆ is A, Q, M, C or E, X₇ is I, E or D and X₈ is F, Y, S or L; (b)X₁X₂X₃X₄X₅X₆HX₇ (SEQ ID NO: 13), in which X₁ is S or T, X₂ is C orabsent, X₃ is T, L or E, X₄ is H or R, X₅ is I, F or E, X₆ is G or V andX₇ is C or N; (c) X₁HPX₂X₃X₄X₅X₆X₇ (SEQ ID NO: 17), in which X₁ is P orW, X₂ is W or E, X₃ is S, A, Q or W, X₄ is M, L, R or P, X₅ is H, F, Wor G, X₆ is V or A and X₇ is I or V; and (d) X₁HX₂X₃X₄X₅ (SEQ ID NO:22), in which X₁ is Y or N, X₂ is E, D or Q, X₃ is T, N, R, M or K, X₄is W, H or F and X₅ is P or G.
 17. A hybridoma capable of producing themonoclonal antibody of claim
 1. 18. A method of obtaining the monoclonalantibody of claim 1, comprising: culturing a hybridoma capable ofproducing the monoclonal antibody so as to produce the monoclonalantibody; and obtaining the monoclonal antibody produced by the culturedhybridoma.
 19. The method of claim 18, further comprising: immunizing ananimal with an antigen comprising an epitope of the Prodefensin-A6protein; obtaining an antibody-producing lymphocyte from the immunizedanimal; and fusing the antibody-producing lymphocyte with a myeloma cellto produce the hybridoma, wherein the epitope is (i) a linear epitopethat includes an amino acid sequence selected from the group consistingof the amino acid sequences of SEQ ID NOS: 4-6, or (ii) a conformationalepitope that includes an amino acid sequence selected from the groupconsisting of the amino acid sequences of SEQ ID NOS: 8-12, 14-16,18-21, and 23-29.